Patent Publication Number: US-2015079633-A1

Title: Production of modified glycoproteins having multiple antennary structures

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a division of U.S. application Ser. No. 12/313,636, filed Nov. 21, 2008, now pending, which is a continuation of U.S. application Ser. No. 10/546,101, filed Aug. 3, 2006, now U.S. Pat. No. 8,697,394, issued Apr. 15, 2014, which is a continuation-in-part of U.S. application Ser. No. 10/680,963, filed Oct. 7, 2003, now U.S. Pat. No. 7,598,055, issued Oct. 6, 2009, which is a continuation-in-part of U.S. application Ser. No. 10/371,877, filed Feb. 20, 2003, now U.S. Pat. No. 7,449,308, issued Nov. 11, 2008, which is a continuation-in-part of U.S. application Ser. No. 09/892,591, filed Jun. 27, 2001, now U.S. Pat. No. 7,029,892, issued Apr. 18, 2006. U.S. application Ser. No. 10/680,963, filed Oct. 7, 2003, is a continuation-in-part of International Application PCT/US02/41510, which claims the benefit of U.S. Provisional No. 60/344,169, filed Dec. 27, 2001, now expired. U.S. application Ser. No. 09/892,591, filed Jun. 27, 2001, claims the benefit of U.S. Provisional No. 60/214,358, filed Jun. 28, 2000, now expired, U.S. Provisional No. 60/215,638, filed Jun. 30, 2000, now expired, and U.S. Provisional No. 60/279,997, filed Mar. 30, 2001. U.S. application Ser. No. 10/500,240, filed Mar. 23, 2005, claims the benefit of U.S. Provisional No. 60/344,169, filed Dec. 27, 2001, now expired. U.S. application Ser. No. 10/546,101, filed Aug. 3, 2006 is a 371 of International PCT/US04/05191, filed Feb. 20, 2004, now U.S. Pat. No. 8,697,394, issued Apr. 15, 2014. 
    
    
     FIELD OF THE INVENTION 
     The present invention is directed to methods and compositions by which non-human eukaryotic host cells, such as fungi or other eukaryotic cells, can be genetically modified to produce glycosylated proteins (glycoproteins) having patterns of glycosylation similar to those of glycoproteins produced by animal cells, especially human cells, which are useful as human or animal therapeutic agents. 
     BACKGROUND OF THE INVENTION 
     Glycosylation Pathways in Humans and Lower Eukaryotes 
     After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins, and if so only in a very unspecific manner (Moens and Vanderleyden (1997)  Arch Microbiol.  168(3):169-175). Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars. The resulting glycan is known as a “high-mannose” type glycan or a mannan. Plant cells and insect cells (such as Sf9 cells) glycosylate proteins in yet another way. By contrast, in higher eukaryotes such as humans, the nascent oligosaccharide side chain may be trimmed to remove several mannose residues and elongated with additional sugar residues that typically are not found in the N-glycans of lower eukaryotes. See, e.g., Bretthauer, et al. (1999)  Biotechnology and Applied Biochemistry  30:193-200; Martinet, et al. (1998)  Biotechnology Letters  20:1171-1177; Weikert, et al. (1999)  Nature Biotechnology,  17:1116-1121; M. Malissard, et al. (2000)  Biochemical and Biophysical Research Communications  267:169-173; Jarvis, et al., (1998)  Current Opinion in Biotechnology  9:528-533; and Takeuchi (1997)  Trends in Glycoscience and Glycotechnology  9:S29-S35. 
     Synthesis of a mammalian-type oligosaccharide structure begins with a set of sequential reactions in the course of which sugar residues are added and removed while the protein moves along the secretory pathway in the host organism. The enzymes which reside along the glycosylation pathway of the host organism or cell determine the resulting glycosylation patterns of secreted proteins. Thus, the resulting glycosylation pattern of proteins expressed in lower eukaryotic host cells differs substantially from the glycosylation pattern of proteins expressed in higher eukaryotes such as humans and other mammals (Bretthauer, 1999). The structure of a typical fungal N-glycan is shown in  FIG. 1A . 
     The early steps of human glycosylation can be divided into at least two different phases: (i) lipid-linked Glc 3 Man 9 GlcNAc 2  oligosaccharides are assembled by a sequential set of reactions at the membrane of the endoplasmic reticulum (ER) ( FIG. 13 ) and (ii) the transfer of this oligosaccharide from the lipid anchor dolichyl pyrophosphate onto de novo synthesized protein. The site of the specific transfer is defined by an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr (SEQ ID NOs:1 and 2) where Xaa can be any amino acid except proline (Gavel and von Heijne (1990)  Protein Eng.  3:433-42). Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the early. Golgi apparatus, where additional mannose residues are removed by Golgi specific alpha (α)-1,2-mannosidases. Processing continues as the protein proceeds through the Golgi. In the medial Golgi, a number of modifying enzymes, including N-acetylglucosaminyl transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV), mannosidase II and fucosyltransferases, add and remove specific sugar residues. Finally, in the trans-Golgi, galactosyltranferases (GalT) and sialyltransferases (ST) produce a glycoprotein structure that is released from the Golgi. It is this structure, characterized by bi-, tri- and tetra-antennary structures, containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid that gives glycoproteins their human characteristics. The structure of a typical human N-glycan is shown in  FIG. 1B . See also  FIGS. 14 and 15  for steps involved in mammalian-type N-glycan processing. 
     In all eukaryotes studied to date, glycoproteins are derived from a common lipid-linked oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -dolichol-pyrophosphate. Within the endoplasmic reticulum, synthesis and processing of dolichol pyrophosphate bound oligosaccharides are identical between all known eukaryotes. However, further processing of the core oligosaccharide by fungal cells, e.g., yeast differs significantly from humans as it moves along the secretory pathway. 
     In yeast, these steps are catalyzed by Golgi residing mannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of human-like proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of  S. cerevisiae , deficient in mannosyltransferase activity (for example och1 or mnn9 mutants) have been shown to be non-lethal and display reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferases, may also have to be eliminated depending on the host&#39;s particular glycosylation pathways. 
     Sugar Nucleotide Precursors 
     The N-glycans of animal glycoproteins typically include galactose, fucose, and terminal sialic acid. These sugars are not found on glycoproteins produced in yeast and filamentous fungi. In humans, the full range of nucleotide sugar precursors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. (Sommers and Hirschberg (1981)  J. Cell Biol.  91(2):A406-A406; Sommers and Hirschberg (1982)  J. Biol. Chem.  257(18):811-817; Perez and Hirschberg (1987)  Methods in Enzymology  138:709-715). 
     Glycosyl transfer reactions typically yield a side product which is a nucleoside diphosphate or monophosphate. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g., GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction is important for efficient glycosylation; for example, GDPase from  Saccharomyces cerevisiae  ( S. cerevisiae ) has been found to be necessary for mannosylation. However that GDPase has 90% reduced activity toward UDP (Berninsone et al. (1994)  J. Biol. Chem.  269(1):207-211). Lower eukaryotes typically lack UDP-specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for Golgi-based glycoprotein synthesis.  Schizosaccharomyces pombe , a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) has been found to have specific UDPase activity, indicating the potential requirement for such an enzyme (Berninsone et al. (1994)  J. Biol. Chem.  269(1):207-211). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product may be important to prevent glycosyl-transferase inhibition in the lumen of the Golgi (Khatara et al. (1974)  Eur. J. Biochem.  44:537-560). See Berninsone et al. (1995)  J. Biol. Chem.  270(24):14564-14567; Beaudet et al. (1998)  Abc Transporters: Biochemical, Cellular, and Molecular Aspects  292: 397-413. 
     Sequential Processing of N-Glycans by Compartmentalized Enzyme Activities 
     Sugar transferases and glycosidases (e.g., mannosidases) line the inner (luminal) surface of the ER and Golgi apparatus and thereby provide a “catalytic” surface that allows for the sequential processing of glycoproteins as they proceed through the ER and Golgi network. The multiple compartments of the cis, medial, and trans Golgi and the trans-Golgi Network (TGN), provide the different localities in which the ordered sequence of glycosylation reactions can take place. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or TGN, it is sequentially exposed to different glucosidases, mannosidases and glycosyltransferases such that a specific carbohydrate structure may be synthesized. Much work has been dedicated to revealing the exact mechanism by which these enzymes are retained and anchored to their respective organelle. The evolving picture is complex but evidence suggests that stem region, membrane spanning region and cytoplasmic tail, individually or in concert, direct enzymes to the membrane of individual organelles and thereby localize the associated catalytic domain to that locus (see, e.g., Gleeson (1998)  Histochem. Cell Biol.  109:517-532). 
     In some cases, these specific interactions were found to function across species. For example, the membrane spanning domain of α2,6-ST from rats, an enzyme known to localize in the trans-Golgi of the animal, was shown to also localize a reporter gene (invertase) in the yeast Golgi (Schwientek et al. (1995)  J. Biol. Chem.  270(10):5483-9). However, the very same membrane spanning domain as part of a full-length α2,6-ST was retained in the ER and not further transported to the Golgi of yeast (Krezdorn et al. (1994)  Eur. J. Biochem.  220(3):809-17). Full length GalT from humans was not even synthesized in yeast, despite demonstrably high transcription levels. In contrast, the transmembrane region of the same human GalT fused to an invertase reporter was able to direct localization to the yeast Golgi, albeit it at low levels. Schwientek and co-workers have shown that fusing 28 amino acids of a yeast mannosyltransferase (MNT1), a region containing a cytoplasmic tail, a transmembrane region and eight amino acids of the stem region, to the catalytic domain of human GalT are sufficient for Golgi localization of an active GalT. Other galactosyltransferases appear to rely on interactions with enzymes resident in particular organelles because, after removal of their transmembrane region, they are still able to localize properly. 
     Improper localization of a glycosylation enzyme may prevent proper functioning of the enzyme in the pathway. For example,  Aspergillus nidulans , which has numerous α-1,2-mannosidases (Eades and Hintz (2000)  Gene  255(1):25-34), does not add GlcNAc to Man 5 GlcNAc 2  when transformed with the rabbit GnTI gene, despite a high overall level of GnTI activity (Kalsner et al. (1995)  Glycoconj. J.  12(3):360-370). GnTI, although actively expressed, may be incorrectly localized such that the enzyme is not in contact with both of its substrates: UDP-GlcNAc and a productive Man 5 GlcNAc 2  substrate (not all Man 5 GlcNAc 2  structures are productive; see below). Alternatively, the host organism may not provide an adequate level of UDP-GlcNAc in the Golgi or the enzyme may be properly localized but nevertheless inactive in its new environment. In addition, Man 5 GlcNAc 2  structures present in the host cell may differ in structure from Man 5 GlcNAc 2  found in mammals. Maras and coworkers found that about one third of the N-glycans from cellobiohydrolase I (CBHI) obtained from  T. reesei  can be trimmed to Man 5 GlcNAc 2  by  A. saitoi  1,2 mannosidase in vitro. Fewer than 1% of those N-glycans, however, could serve as a productive substrate for GnTI. Maras et al. (1997)  Eur. J. Biochem.  249:701-707. The mere presence of Man 5 GlcNAc 2 , therefore, does not assure that further in vivo processing of Man 5 GlcNAc 2  can be achieved. It is formation of a productive, GnTI-reactive Man 5 GlcNAc 2  structure that is required. Although Man 5 GlcNAc 2  could be produced in the cell (about 27 mol %), only a small fraction could be converted to Man 5 GlcNAc 2  (less than about 5%, see Chiba et al. WO 01/14522). 
     To date, there is no reliable way of predicting whether a particular heterologously expressed glycosyltransferase or mannosidase in a lower eukaryote will be (1), sufficiently translated (2), catalytically active or (3) located to the proper organelle within the secretory pathway. Because all three of these are necessary to affect glycosylation patterns in lower eukaryotes, a systematic scheme to achieve the desired catalytic function and proper retention of enzymes in the absence of predictive tools, which are currently not available, would be desirable. 
     Production of Therapeutic Glycoproteins 
     A significant number of proteins isolated from humans or animals are post-translationally modified, with glycosylation being one of the most significant modifications. An estimated 70% of all therapeutic proteins are glycosylated and thus currently rely on a production system (i.e., host cell) that is able to glycosylate in a manner similar to humans. Several studies have shown that glycosylation plays an important role in determining the (1) immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4) efficacy of therapeutic proteins. It is thus not surprising that substantial efforts by the pharmaceutical industry have been directed at developing processes to obtain glycoproteins that are as “humanoid” or “human-like” as possible. To date, most glycoproteins are made in a mammalian host system. This may involve the genetic engineering of such mammalian cells to enhance the degree of sialylation (i.e., terminal addition of sialic acid) of proteins expressed by the cells, which is known to improve pharmacokinetic properties of such proteins. Alternatively, one may improve the degree of sialylation by in vitro addition of such sugars using known glycosyltransferases and their respective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialic acid). 
     While most higher eukaryotes carry out glycosylation reactions that are similar to those found in humans, recombinant human proteins expressed in the above mentioned host systems invariably differ from their “natural” human counterpart (Raju et al. (2000)  Glycobiology  10(5): 477-486). Extensive development work has thus been directed at finding ways to improve the “human character” of proteins made in these expression systems. This includes the optimization of fermentation conditions and the genetic modification of protein expression hosts by introducing genes encoding enzymes involved in the formation of human-like glycoforms. Goochee et al. (1999)  Biotechnology  9(12):1347-55; Andersen and Goochee (1994)  Curr Opin Biotechnol.  5(5):546-49; Werner et al. (1998)  Arzneimittelforschung.  48(8):870-80; Weikert et al. (1999)  Nat Biotechnol.  17(11):1116-21; Yang and Butler (2000)  Biotech. Bioeng.  68:370-80. Inherent problems associated with all mammalian expression systems have not been solved. 
     Glycoprotein Production Using Eukaryotic Microorganisms 
     Although the core oligosaccharide structure transferred to a protein in the endoplasmic reticulum is basically identical in mammals and lower eukaryotes, substantial differences have been found in the subsequent processing reactions which occur in the Golgi apparatus of fungi and mammals. In fact, even amongst different lower eukaryotes there exist a great variety of glycosylation structures. This has historically prevented the use of lower eukaryotes as hosts for the production of recombinant human glycoproteins despite otherwise notable advantages over mammalian expression systems. 
     Therapeutic glycoproteins produced in a microorganism host such as yeast utilizing the endogenous host glycosylation pathway differ structurally from those produced in mammalian cells and typically show greatly reduced therapeutic efficacy. Such glycoproteins are typically immunogenic in humans and show a reduced half-life (and thus bioactivity) in vivo after administration (Takeuchi (1997)  Trends in Glycoscience and Glycotechnology  9:S29-S35). Specific receptors in humans and animals (i.e., macrophage mannose receptors) can recognize terminal mannose residues and promote the rapid clearance of the foreign glycoprotein from the bloodstream. Additional adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. 
     Yeast and filamentous fungi have both been successfully used for the production of recombinant proteins, both intracellular and secreted (Cereghino and Cregg (2000)  FEMS Microbiology Reviews  24(1):45-66; Harkki et al. (1989)  Bio - Technology  7(6):596; Berka et al. (1992)  Abstr. Papers Amer. Chem. Soc.  203:121-BIOT; Svetina et al. (2000)  J. Biotechnol.  76(2-3):245-251). Various yeasts, such as  K. lactis, Pichia pastoris, Pichia methanolica , and  Hansenula polymorpha , have played particularly important roles as eukaryotic expression systems because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as  Aspergillus niger, Fusarium  sp,  Neurospora crassa  and others, have been used to efficiently produce glycoproteins at the industrial scale. However, as noted above, glycoproteins expressed in any of these eukaryotic microorganisms differ substantially in N-glycan structure from those in animals. This has prevented the use of yeast or filamentous fungi as hosts for the production of many therapeutic glycoproteins. 
     Although glycosylation in yeast and fungi is very different than in humans, some common elements are shared. The first step, the transfer of the core oligosaccharide structure to the nascent protein, is highly conserved in all eukaryotes including yeast, fungi, plants and humans (compare  FIGS. 1A and 1B ). Subsequent processing of the core oligosaccharide, however, differs significantly in yeast and involves the addition of several mannose sugars. This step is catalyzed by mannosyltransferases residing in the Golgi (e.g., OCH1, MNT1, MNN1, etc.), which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of humanoid proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of  S. cerevisiae  deficient in mannosyltransferase activity (e.g., och1 or mnn9 mutants) have shown to be non-lethal and display a reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferase, may also have to be eliminated depending on the host&#39;s particular endogenous glycosylation pattern. After reducing undesired endogenous glycosylation reactions, the formation of complex N-glycans has to be engineered into the host system. This requires the stable expression of several enzymes and sugar-nucleotide transporters. Moreover, one has to localize these enzymes so that a sequential processing of the maturing glycosylation structure is ensured. 
     Several efforts have been made to modify the glycosylation pathways of eukaryotic microorganisms to provide glycoproteins more suitable for use as mammalian therapeutic agents. For example, several glycosyltransferases have been separately cloned and expressed in  S. cerevisiae  (GalT, GnTI),  Aspergillus nidulans  (GnTI) and other fungi (Yoshida et al. (1999)  Glycobiology  9(1):53-8, Kalsner et al. (1995)  Glycoconj. J.  12(3):360-370). However, N-glycans resembling those made in human cells were not obtained. 
     Yeasts produce a variety of mannosyltransferases (e.g., 1,3-mannosyltransferases such as MNN1 in  S. cerevisiae ; Graham and Emr (1991)  J. Cell. Biol.  114(2):207-218), 1,2-mannosyltransferases (e.g., KTR/KRE family from  S. cerevisiae ), 1,6-mannosyltransferases (e.g., OCH1 from  S. cerevisiae ), mannosylphosphate transferases and their regulators (e.g., MNN4 and MNN6 from  S. cerevisiae ) and additional enzymes that are involved in endogenous glycosylation reactions. Many of these genes have been deleted individually giving rise to viable organisms having altered glycosylation profiles. Examples are shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Examples of yeast strains having altered mannosylation 
               
            
           
           
               
               
               
               
               
            
               
                 Strain 
                 N-glycan (wild type) 
                 Mutation 
                 N-glycan (mutant) 
                 Reference 
               
               
                   
               
               
                 
                   S. pombe 
                 
                 Man &gt;9 GlcNAc 2   
                 OCH1 
                 Man 8 GlcNAc 2   
                 Yoko-o et al. 
               
               
                   
                   
                   
                   
                 (2001)  FEBS Lett.   
               
               
                   
                   
                   
                   
                 489(1): 75-80 
               
               
                 
                   S. cerevisiae 
                 
                 Man &gt;9 GlcNAc 2   
                 OCH1/MNN1 
                 Man 8 GlcNAc 2   
                 Nakanishi-Shindo 
               
               
                   
                   
                   
                   
                 et al. (1993) 
               
               
                   
                   
                   
                   
                 
                   J. Biol. Chem. 
                 
               
               
                   
                   
                   
                   
                 268(35): 26338- 
               
               
                   
                   
                   
                   
                 26345 
               
               
                 
                   S. cerevisiae 
                 
                 Man &gt;9 GlcNAc 2   
                 OCH1/MNN1/MNN4 
                 Man 8 GlcNAc 2   
                 Chiba et al. (1998) 
               
               
                   
                   
                   
                   
                 
                   J. Biol. Chem. 
                 
               
               
                   
                   
                   
                   
                 273, 26298-26304 
               
               
                 
                   P. pastoris 
                 
                 Hyperglycosylated 
                 OCH1 (complete 
                 Not 
                 Welfide, Japanese 
               
               
                   
                   
                 deletion) 
                 hyperglycosylated 
                 Application 
               
               
                   
                   
                   
                   
                 Publication No. 
               
               
                   
                   
                   
                   
                 8-336387 
               
               
                 
                   P. pastoris 
                 
                 Man &gt;8 GlcNAc 2   
                 OCH1 (disruption) 
                 Man &gt;8 GlcNAc 2   
                 Contreras et al. 
               
               
                   
                   
                   
                   
                 WO 02/00856 A2 
               
               
                   
               
            
           
         
       
     
     Japanese Patent Application Publication No. 8-336387 discloses the deletion of an OCH1 homolog in  Pichia pastoris . In  S. cerevisiae , OCH1 encodes a 1,6-mannosyltransferase, which adds a mannose to the glycan structure Man 8 GlcNAc 2  to yield Man 9 GlcNAc 2 . The Man 9 GlcNAc 2  structure, which contains three 1,6 mannose residues, is then a substrate for further 1,2-, 1,6-, and 1,3-mannosyltransferases in vivo, leading to the hypermannosylated glycoproteins that are characteristic for  S. cerevisiae  and which typically may have 30-40 mannose residues per N-glycan. Because the Och1p initiates the transfer of 1,6 mannose to the Man 8 GlcNAc 2  core, it is often referred to as the “initiating 1,6 mannosyltransferase” to distinguish it from other 1,6 mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4 mutant strain of  S. cerevisiae , proteins glycosylated with Man 8 GlcNAc 2  accumulate and hypermannosylation does not occur. However, Man 8 GlcNAc 2  is not a substrate for mammalian glycosyltransferases, such as human UDP-GlcNAc transferase I, and accordingly, the use of that mutant strain, in itself, is not useful for producing mammalian-like proteins, i.e., those with complex or hybrid glycosylation patterns. 
     One can trim Man 8 GlcNAc 2  structures to a Man 5 GlcNAc 2  isomer in  S. cerevisiae  (although high efficiency trimming greater than 50% in vivo has yet to be demonstrated) by engineering a fungal mannosidase from  A. saitoi  into the endoplasmic reticulum (ER). The shortcomings of this approach are two-fold: (1) it is not clear whether the Man 5 GlcNAc 2  structures formed are in fact formed in vivo (rather than having been secreted and further modified by mannosidases outside the cell); and (2) it is not clear whether any Man 5 GlcNAc 2  structures formed, if in fact formed in vivo, are the correct isoform to be a productive substrate for subsequent N-glycan modification by GlcNAc transferase I (Maras et al. (1997)  Eur. J. Biochem.  249:701-707). 
     With the objective of providing a more human-like glycoprotein derived from a fungal host, U.S. Pat. No. 5,834,251 discloses a method for producing a hybrid glycoprotein derived from  Trichoderma reesei . A hybrid N-glycan has only mannose residues on the Manα1-6 arm of the core mannose structure and one or two complex antennae on the Manα1-3 arm. While this structure has utility, the method has the disadvantage that numerous enzymatic steps must be performed in vitro, which is costly and time-consuming. Isolated enzymes are expensive to prepare and need costly substrates (e.g., UDP-GlcNAc). The method also does not allow for the production of complex glycans on a desired protein. 
     Intracellular Mannosidase Activity Involved in N-glycan Trimming 
     Alpha-1,2-mannosidase activity is required for the trimming of Man 8 GlcNAc 2  to form Man 5 GlcNAc 2 , which is a major intermediate for complex N-glycan formation in mammals. Previous work has shown that truncated murine, fungal and human α-1,2-mannosidase can be expressed in the methylotropic yeast  P. pastoris  and display Man 8 GlcNAc 2  to Man 5 GlcNAc 2  trimming activity (Lal et al. (1998)  Glycobiology  8(10):981-95; Tremblay et al. (1998)  Glycobiology  8(6):585-95, Callewaert et al. (2001)  FEBS Lett.  503(2-3):173-8). However, to date, no reports exist that show the high level in vivo trimming of Man 8 GlcNAc 2  to Man 5 GlcNAc 2  on a secreted glycoprotein from  P. pastoris.    
     Moreover, the mere presence of an α-1,2-mannosidase in the cell does not, by itself, ensure proper intracellular trimming of Man 8 GlcNAc 2  to Man 5 GlcNAc 2 . (See, e.g., Contreras et al. WO 02/00856 A2, in which an HDEL tagged mannosidase of  T. reesei  is localized primarily in the ER and co-expressed with an influenza haemagglutinin (HA) reporter protein on which virtually no Man 5 GlcNAc 2  could be detected. See also Chiba et al. (1998)  J. Biol. Chem.  273(41): 26298-26304, in which a chimeric α-1,2-mannosidase/Och1p transmembrane domain fusion localized in the ER, early Golgi and cytosol of  S. cerevisiae , had no mannosidase trimming activity). Accordingly, mere localization of a mannosidase in the ER or Golgi is insufficient to ensure activity of the respective enzyme in that targeted organelle. (See also, Martinet et al. (1998)  Biotech. Letters  20(12): 1171-1177, showing that α-1,2-mannosidase from  T. reesei , while localizing intracellularly, increased rather than decreased the extent of mannosylation). To date, there is no report that demonstrates the intracellular localization of an active heterologous α-1,2-mannosidase in either yeast or fungi using a transmembrane localization sequence. 
     While it is useful to engineer strains that are able to produce Man 5 GlcNAc 2  as the primary N-glycan structure, any attempt to further modify these high mannose precursor structures to more closely resemble human glycans requires additional in vivo or in vitro steps. Methods to further humanize glycans from fungal and yeast sources in vitro are described in U.S. Pat. No. 5,834,251 (supra). If Man 5 GlcNAc 2  is to be further humanized in vivo, one has to ensure that the generated Man 5 GlcNAc 2  structures are, in fact, generated intracellularly and not the product of mannosidase activity in the medium. Complex N-glycan formation in yeast or fungi will require high levels of Man 5 GlcNAc 2  to be generated within the cell because only intracellular Man 5 GlcNAc 2  glycans can be further processed to hybrid and complex N-glycans in vivo. In addition, one has to demonstrate that the majority of Man 5 GlcNAc 2  structures generated are in fact a substrate for GnTI and thus allow the formation of hybrid and complex N-glycans. 
     Accordingly, the need exists for methods to produce glycoproteins characterized by a high intracellular Man 5 GlcNAc 2  content which can be further processed into human-like glycoprotein structures in non-human eukaryotic host cells, and particularly in yeast and filamentous fungi. 
     N-Acetylglucosaminyltransferases 
     N-Acetylglucosaminyltransferases (“GnTs”) belong to another class of glycosylation enzymes that modify N-linked oligosaccharides in the secretory pathway. Such glycosyltransferases catalyze the transfer of a monosaccharide from specific sugar nucleotide donors onto particular hydroxyl position of a monosaccharide in a growing glycan chain in one of two possible anomeric linkages (either α or β). Dennis et al. (1999)  Bioessays  21(5):412-21. Specific GnTs add N-acetylglucosamine (“GlcNAc”) onto the Manα1,6 arm or the Manα1,3 arm of an N-glycan substrate (e.g., Man 5 GlcNAc 2  (“mannose-5 core”) and Man 3 GlcNAc 2  (an “inner core structure”)). The reaction product (e.g., GlcNAcMan 5 GlcNAc 2  or GlcNAc 2 Man 3 GlcNAc 2 ) can then be modified into bi-, tri-, tetra- and penta-antennary N-linked oligosaccharide structures. N-Acetylglucosaminyltransferase III (“GnTIII”) is an enzyme that catalyzes the addition of a GlcNAc, on the middle mannose of the trimannose core (Manα1,6 (Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn) of an N-linked oligosaccharide. The addition by GnTIII of a bisecting GlcNAc to an acceptor substrate (e.g. trimannose core) yields a so-called bisected N-glycan. For example, the addition by GnTIII of a bisecting GlcNAc to the GlcNAcMan 3 GlcNAc 2  structure may yield a bisected N-glycan, GlcNAc 2 Man 3 GlcNAc 2 . Similarly, the addition by GnTIII of a bisecting GlcNAc to a GlcNAc 2 Man 3 GlcNAc 2  structure yields another bisected N-glycan, GlcNAc 3 Man 3 GlcNAc 2 . This latter structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC). Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80. Other bisected N-glycans can be formed by the action of GnTIII. For example, GlcNAcMan 4 GlcNAc 2  can be converted to bisected GlcNAc 2 Man 4 GlcNAc 2 , Man 5 GlcNAc 2  can be converted to bisected GlcNAcMan 5 GlcNAc 2 , and GlcNAcMan 5 GlcNAc 2  can be converted to bisected GlcNAc 2 Man 5 GlcNAc 2 . See, e.g., Narasimhan (1982)  J. Biol. Chem.  257:10235-42. Thus far, GnTIII activity has only been shown in mammalian cells. 
     Re-engineering glycoforms of immunoglobulins expressed by mammalian cells is a tedious and cumbersome task. Especially in the case of GnTIII, where over-expression of this enzyme has been implicated in growth inhibition, methods involving regulated (inducible) gene expression had to be employed to produce immunoglobulins with bisected N-glycans. Umana et al. (1999)  Biotechnol Bioeng.  65(5):542-9; Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80; Umana et al. WO 03/011878; U.S. Pat. No. 6,602,684. Such a growth-inhibition effect complicates the ability to coexpress the target protein and GnTIII and may impose an upper limit on GnTIII overexpression. U.S. Pat. No. 6,602,684. Careful optimization of the expression levels of GnTIII may be necessary. Id. As described above, however, development of the lower eukaryotic host cells used in such a protein production system requires that the endogenous glycosylation pathways of the host cells be further modified. 
     The enzymes GnTIV, GnTV and GnTIX expressed in mammalian cells are known to catalyze the transfer of GlcNAc residues in particular conformation onto oligosaccharide substrates producing multiantennary glycan structures. UDP-N-acetylglucosamine:α1,3- D -mannoside β1,4-N-acetylglucosaminyl-transferase (GnTIV; EC 2.4.1.145) catalyzes the transfer of GlcNAc from UDP-GlcNAc in residues in β1,4 linkage to α1,3- D -mannoside on GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (Gleeson and Schachter,  J Biol Chem.  1983 May 25; 258(10):6162-73; Schachter et al., (1989)  Methods Enzymol.,  179, 351-397). UDP-N-acetylglucosamine:α-6- D -mannoside β1,6-N-acetylglucosaminyltransferase (GnTV; EC 4.1.155) catalyzes the addition of an N-acetylglucosamine to the α1,6 mannosyl core in a β1,6 linkage forming tri- and tetraantennary N-glycans. 
     Similarly, the expression of GnTVI in avian cells catalyzes the transfer of GlcNAc residues onto oligosaccharide substrates. Specifically, UDP N-acetyl- D -glucosamine (GlcNAc):GlcNAcβ1-6(GlcNAc β1-2)Manα1-R[GlcNAc to Man]β1,4-N-acetylglucosaminyltransferase VI(GnTVI) catalyzes the formation of pentaantennary N-glycans (Sakamoto et al., J Biol Chem. 2000 Nov. 17; 275(46):36029-34). The gene encoding GnTVI have been purified and isolated recently. Taniguchi et al., JP2002209587A2. 
     Substrates required to produce complex multiantennary structures have not been synthesized in fungal hosts until recently (Hamilton et al., Science. 2003 Aug. 29; 301 (5637): 1244-6). Mammalian cells typically produce an array of complex glycans such as biantennary, triantennary, tetraantennary and even pentaantennary glycoforms through sequential reaction of specific GnTs. In the Golgi apparatus of such cells, N-glycan processing of glycoproteins produces biantennary structures predominantly, in addition to the formation of triantennary and tetraantennary structures. It is currently understood that in the formation of complex glycans specific GnTs catalyze specific β-GlcNAc linkages (e.g., β1,2; β1,4; β1,6), producing multiantennary glycans in mammalian cells. A major shortcoming of these cells, however, is that they are incapable of producing any one homogenous glycoform in high yield. 
     Recently, lower eukaryotes have been engineered to produce complex glycans in homogeneous forms at significant levels (Science. 2003 Aug. 29; 301(5637):1244-6). The ability to produce multiantennary complex glycans in lower eukaryotes would provide large amounts of properly folded and glycosylated proteins on an industrial scale at low cost, in faster time, safer and in higher quality. What is needed, therefore, is a protein production system utilizing the inherent capability of robust product titers such as those produced in lower eukaryotic host cells (e.g. yeast and filamentous fungi), which is capable of producing multiantennary (and optionally, bisected) N-linked glycans on proteins, especially therapeutic proteins, expressed in these cells. 
     SUMMARY OF THE INVENTION 
     Host cells and cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions which mimic the processing of glycoproteins in mammals, especially in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their mammalian, e.g., human counterparts. Host cells of the invention, e.g., lower eukaryotic microorganisms and other non-human, eukaryotic host cells grown in culture, are modified to produce N-glycans, such as bisected N-glycans, or other structures produced along human glycosylation pathways. This result is achieved using a combination of engineering and/or selection of strains that do not, for example, express enzymes that create the undesirable structures characteristic of the fungal glycoproteins and that do, for example, express heterologous enzymes capable of producing a “human-like” glycoprotein. 
     The present invention thus provides a glycoprotein production method using (1) a lower eukaryotic host such as a unicellular or filamentous fungus, or (2) any non-human eukaryotic organism that has a different glycosylation pattern from humans, to modify the glycosylation composition and structures of the proteins made in a host organism (“host cell”) so that they resemble more closely carbohydrate structures found in mammalian, e.g., human proteins. The process allows one to obtain an engineered host cell which can be used to express and target any desirable gene(s), e.g., one involved in glycosylation, by methods that are well-established in the scientific literature and generally known to the artisan in the field of protein expression. Host cells with modified oligosaccharides are created or selected. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained. 
     Accordingly, in one embodiment, the invention provides methods for making a human-like glycoprotein in a lower eukaryotic host cell by introduction into the cell of an N-acetylglucosaminyltransferase III activity. In a preferred embodiment, the N-acetylglucosaminyltransferase III activity is expressed in the cell, and in an even more preferred embodiment, this expression results in the production of N-glycans comprising GlcNAc 3 Man 3 GlcNAc 2 , GlcNAc 2 Man 3 GlcNAc 2  or GlcNAc 2 Man 5 GlcNAc 2  bisected structures. In another preferred embodiment, the N-acetylglucosaminyltransferase III activity is substantially intracellular. In another preferred embodiment of the invention, the glycoprotein including the N-glycans with bisected structures is isolated from the lower eukaryotic host cell. In an even more preferred embodiment, the glycoprotein produced in the host cell is a therapeutic protein. 
     In another aspect, the invention provides a lower eukaryotic host cell that includes both an N-acetylglucosaminyltransferase III activity and an N-acetylglucosaminyltransferase II activity. In a preferred embodiment, the host cell including the N-acetylglucosaminyltransferase III activity produces N-glycans comprising GlcNAcMan 3 GlcNAc 2  structures that are capable of reacting with this activity. In a more preferred embodiment, the activity produces a bisected glycan. The lower eukaryotic host cell of some embodiments of the invention may thus include an N-glycan with a bisected glycan. In a preferred embodiment, the N-glycan includes greater than 10 mole % of the bisected glycan. In some embodiments, the host cell includes an N-glycan that comprises GlcNAc 3 Man 3 GlcNAc 2 , GlcNAc 2 Man 3 GlcNAc 2 , or GlcNAc 2 Man 5 GlcNAc 2  bisected structures. In a preferred embodiment, the host cell includes a Man 5 GlcNAc 2  core structure or a Man 3 GlcNAc 2  core structure that is modified by a bisecting GlcNAc. In an even more preferred embodiment, the cell produces greater than 10 mole % of the modified structure. 
     In another embodiment of the invention, the lower eukaryotic host cell contains an N-acetylglucosaminyltransferase I activity in addition to the N-acetylglucosaminyltransferase III activity. In a preferred embodiment, the activities are substantially intracellular. In another preferred embodiment, the cell produces N-glycans comprising GlcNAcMan 3 GlcNAc 2  that are capable of reacting with the GnTIII activity. In an even more preferred embodiment, the GnTIII activity of the cell produces a bisected glycan. 
     In another embodiment, the lower eukaryotic host cell of the invention contains both an N-acetylglucosaminyltransferase III activity and mannosidase II activity. In a preferred embodiment, the host cell further contains an N-acetylglucosaminyltransferase I activity. In another preferred embodiment, the host cell further contains an N-acetylglucosaminyltransferase II activity. In another preferred embodiment, the host cell further contains both an N-acetylglucosaminyltransferase I activity and an N-acetylglucosaminyltransferase II activity. 
     The present invention also provides methods for making a human-like glycoprotein in a lower eukaryotic host cell by introduction into the cell of an N-acetylglucosaminyltransferase VI activity. In a preferred embodiment, the N-acetylglucosaminyltransferase VI activity is expressed in the cell, and in an even more preferred embodiment, this expression results in the production of N-glycans comprising GlcNAc 3 Man 3 GlcNAc 2  structure. In another preferred embodiment the N-acetylglucosaminyltransferase VI activity is substantially intracellular. In another preferred embodiment of the invention, the glycoprotein including the N-glycans with triantennary structures is isolated from the lower eukaryotic host cell. In a more preferred embodiment, the N-glycan includes greater than 90 mole % of the triantennary glycan. In an even more preferred embodiment, the glycoprotein produced in the host cell is a therapeutic protein. 
     Thus, in another aspect, the invention provides a lower eukaryotic host cell that includes both an N-acetylglucosaminyltransferase IV activity and an N-acetylglucosaminyltransferase V activity. In a preferred embodiment, the host cell including the N-acetylglucosaminyltransferase IV and V activities produces N-glycans comprising GlcNAc 4 Man 3 GlcNAc 2  structures. In a more preferred embodiment, the activity produces a tetraantennary glycan. The lower eukaryotic host cell of some embodiments of the invention may thus include an N-glycan with a tetraantennary glycan. In some embodiments, the host cell includes an N-glycan that comprises GlcNAc 3 Man 3 GlcNAc 2  and GlcNAc 4 Man 3 GlcNAc 2  structures. In a preferred embodiment, the host cell includes a GlcNAcMan 5 GlcNAc 2  core structure that is modified by a GnT IV or a GlcNAc 2 Man 3 GlcNAc 2  core structure that is modified by a GnT IV and GnT V. In a preferred embodiment, the N-glycan includes greater than 70 mole % of the tetraantennary glycan. In an even more preferred embodiment, the cell produces greater than 75 mole % of tetra-antennary glycans. 
     The present invention also provides a lower eukaryotic host cell that includes an N-acetylglucosaminyltransferase VI activity. In a preferred embodiment, the host cell expressing the N-acetylglucosaminyltransferase VI activity produces N-glycans comprising GlcNAc 5 Man 3 GlcNAc 2  structures (e.g., pentaantennary glycans). The lower eukaryotic host cell of some embodiments of the invention may thus include an N-glycan with a pentaantennary glycan. In some embodiments, the host cell includes an N-glycan that comprises GlcNAc 5 Man 3 GlcNAc 2  structures. In a preferred embodiment, the host cell includes a GlcNAc 2 Man 3 GlcNAc 2  core structure that is modified by a GnT VI. 
     In another embodiment, the invention provides methods for making a human-like glycoprotein in a lower eukaryotic host cell by introduction into the cell of an N-acetylglucosaminyltransferase IX activity. In a preferred embodiment, the N-acetylglucosaminyltransferase IX activity is expressed in the cell, and in an even more preferred embodiment, this expression results in the production of N-glycans comprising GlcNAc 3 Man 3 GlcNAc 2  and GlcNAc 4 Man 3 GlcNAc 2  structures. In another preferred embodiment, the N-acetylglucosaminyltransferase IX activity is substantially intracellular. In another preferred embodiment of the invention, the glycoprotein including the N-glycans with multiantennary structures is isolated from the lower eukaryotic host cell. In an even more preferred embodiment, the glycoprotein produced in the host cell is a therapeutic protein. 
     In another embodiment of the invention, the lower eukaryotic host cell contains an N-acetylglucosaminyltransferase I activity and an N-acetylglucosaminyltransferase II activity. In a preferred embodiment, the activities are substantially intracellular. In another preferred embodiment, the cell produces N-glycans comprising GlcNAc 2 Man 3 GlcNAc 2  that are capable of reacting with the GnTIV activity producing triantennary glycans. In an even more preferred embodiment, the GnTV activity of the cell produces a tetraantennary glycans. 
     In another embodiment, the lower eukaryotic host cell of the invention contains both an N-acetylglucosaminyltransferase IV activity and a mannosidase II activity. In a preferred embodiment, the host cell further contains an N-acetylglucosaminyltransferase I activity. In another preferred embodiment, the host cell further contains an N-acetylglucosaminyltransferase II activity. In another preferred embodiment, the host cell further contains an N-acetylglucosaminyltransferase V activity. 
     In certain preferred embodiments, the host cell of the invention is deficient in an OCH1 mannosyltransferase activity. Such a cell may, for example, be deficient in a Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity. In yet another embodiment, the host cell of the invention may further comprise an α-1,2-mannosidase I activity. In another embodiment, the host cell may further comprise a sugar nucleotide transporter. Preferably, the host cell comprises a UDP-GlcNAc transporter wherein the transfer of GlcNAc residues is facilitated by any one of the above mentioned N-acetylglucosaminyltransferase activities. 
     The present invention also provides glycoproteins that are made by the processes of the invention. In one embodiment, the glycoprotein includes a bisecting GlcNAc on a Man 5 GlcNAc 2  or Man 3 GlcNAc 2  core structure and is produced in a lower eukaryotic host cell. In another embodiment, the glycoprotein includes a bisecting GlcNAc attached to a Man 5 GlcNAc 2 , Man 4 GlcNAc 2 , Man 3 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , or a GlcNAc 2 Man 3 GlcNAc 2  core structure and is produced in a lower eukaryotic host cell. In a preferred embodiment, greater than 10 mole % of the core structures of the glycoprotein of the invention are modified by the bisecting GlcNAc. 
     In yet another embodiment, the invention provides a glycoprotein that includes a triantennary structure such as GlcNAc 3 Man 3 GlcNAc 2  and is produced in a lower eukaryotic host cell. In a preferred embodiment, greater than 90 mole % of the core structures of the glycoprotein of the invention are modified by the GnTIV. In another embodiment, the glycoprotein includes a tetraantennary structure such as GlcNAc 4 Man 3 GlcNAc 2  and is produced in a lower eukaryotic host cell. In a more preferred embodiment, greater than 75 mole % of the core structures of the glycoprotein of the invention are modified by the GnTV. 
     In another aspect, the invention provides pharmaceutical compositions that contain the human-like glycoproteins produced in a lower eukaryotic host cell. Also provided according to the invention are vectors encoding proteins having one or more N-acetylglucosaminyltransferase III, IV, V, VI and IX activities and containing attached targeting peptide sequences. In a preferred embodiment, the proteins encoded by the vectors are localized in a lower eukaryotic host cell such that they produce N-glycans having bisected and/or multiantennary structures. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a schematic diagram of a typical fungal N-glycosylation pathway. 
         FIG. 1B  is a schematic diagram of a typical human N-glycosylation pathway. 
         FIG. 2  depicts construction of a combinatorial DNA library of fusion constructs. Panel A diagrams the insertion of a targeting peptide fragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). Panel B shows the generated targeting peptide sub-library having restriction sites NotI-AscI. Panel C diagrams the insertion of a catalytic domain region into pJN347, a modified pUC19 vector. Panel D shows the generated catalytic domain sub-library having restriction sites NotI, AscI and PacI. Panel E depicts one particular fusion construct generated from the targeting peptide sub-library and the catalytic domain sub-library. 
         FIGS. 3A and 3B  illustrate the  M. musculus  α-1,2-mannosidase IA open reading frame nucleic acid sequence (SEQ ID NO:48) and encoded polypeptide sequence (SEQ ID NO:49). The sequences of the PCR primers used to generate N-terminal truncations are underlined. 
         FIG. 4A-4F  illustrate engineering of vectors with multiple auxotrophic markers and genetic integration of target proteins in the  P. pastoris  OCH1 locus. 
         FIGS. 5A-5E  show MALDI-TOF analysis demonstrating production of kringle 3 domain of human plasminogen (K3) glycoproteins having Man 5 GlcNAc 2  as the predominant N-glycan structure in  P. pastoris .  FIG. 5A  depicts the standard Man 5 GlcNAc 2  [a] glycan (Glyko, Novato, Calif.) and Man 5 GlcNAc 2 +Na + [b].  FIG. 5B  shows PNGase-released glycans from K3 wild type. The N-glycans shown are as follows: Man 9 GlcNAc 2  [d]; Man 10 GlcNAc 2  [e]; Man 11 GlcNAc 2  [f]; Man 12 GlcNAc 2  [g].  FIG. 5C  depicts the och1 deletion resulting in the production of Man 8 GlcNAc 2  [c] as the predominant N-glycan.  FIGS. 5D and 5E  show the production of Man 5 GlcNAc 2  [b] after in vivo trimming of Man 8 GlcNAc 2  with a chimeric α-1,2-mannosidase. The predominant N-glycan is indicated by a peak with a mass (m/z) of 1253 consistent with its identification as Man 5 GlcNAc 2  [b]. 
         FIGS. 6A-6F  show MALDI-TOF analysis demonstrating production of IFN-β glycoproteins having Man 5 GlcNAc 2  as the predominant N-glycan structure in  P. pastoris .  FIG. 6A  shows the standard Man 5 GlcNAc 2  [a] and Man 5 GlcNAc 2 +Na + [b] as the standard (Glyko, Novato, Calif.).  FIG. 6B  shows PNGase-released glycans from IFN-β wildtype.  FIG. 6C  depicts the och1 knock-out producing Man 8 GlcNAc 2  [c]; Man 9 GlcNAc 2  [d]; Man 10 GlcNAc 2  [e]; Man 11 GlcNAc 2  [f]; Man 12 GlcNAc 2  [g]; and no production of Man 5 GlcNAc 2  [b].  FIG. 6D  shows relatively small amount of Man 5 GlcNAc 2  [b] among other intermediate N-glycans Man 8 GlcNAc 2  [c] to Man 12 GlcNAc 2 [g].  FIG. 6E  shows a significant amount of Man 5 GlcNAc 2  [b] relative to the other glycans Man 8 GlcNAc 2  [c] and Man 9 GlcNAc 2  [d] produced by pGC5 ( Saccharomyces  MNS1(m)/mouse mannosidase IB Δ99).  FIG. 6F  shows predominant production of Man 5 GlcNAc 2  [b] on the secreted glycoprotein IFN-β by pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187). The N-glycan is indicated by a peak with a mass (m/z) of 1254 consistent with its identification as Man 5 GlcNAc 2  [b]. 
         FIGS. 7A-7C  show a high performance liquid chromatogram for: (A) Man 9 GlcNAc 2  standard labeled with 2-AB (negative control); (B) supernatant of medium  P. pastoris , Δoch1 transformed with pFB8 mannosidase, which demonstrates a lack of extracellular mannosidase activity in the supernatant; and (C) Man 9 GlcNAc 2  standard labeled with 2-AB after exposure to  T. reesei  mannosidase (positive control). 
         FIGS. 8A-8C  show a high performance liquid chromatogram for: (A) Man 9 GlcNAc 2  standard labeled with 2-AB (negative control); (B) supernatant of medium  P. pastoris , Δoch1 transformed with pGC5 mannosidase, which demonstrates a lack of extracellular mannosidase activity in the supernatant; and (C) Man 9 GlcNAc 2  standard labeled with 2-AB after exposure to  T. reesei  mannosidase (positive control). 
         FIGS. 9A-9C  show a high performance liquid chromatogram for: (A) Man 9 GlcNAc 2  standard labeled with 2-AB (negative control); (B) supernatant of medium  P. pastoris , Δoch1 transformed with pBC18-5 mannosidase, which demonstrates lack of extracellular mannosidase activity in the supernatant; and (C) supernatant of medium  P. pastoris , Δoch1 transformed with pDD28-3, which demonstrates activity in the supernatant (positive control). 
         FIGS. 10A-10B  demonstrate the activity of an UDP-GlcNAc transporter in the production of GlcNAcMan 5 GlcNAc 2  in  P. pastoris .  FIG. 10A  depicts a  P. pastoris  strain (YSH-3) with a human GnTI but without the UDP-GlcNAc transporter resulting in some production of GlcNAcMan 5 GlcNAc 2  [b] but a predominant production of Man 5 GlcNAc 2  [a].  FIG. 10B  depicts the addition of UDP-GlcNAc transporter from  K. lactis  in a strain (PBP-3) with the human GnTI, which resulted in the predominant production of GlcNAcMan 5 GlcNAc 2  [b]. The single prominent peak of mass (m/z) at 1457 is consistent with its identification as GlcNAcMan 5 GlcNAc 2  [b] as shown in  FIG. 10B . 
         FIG. 11  shows a pH optimum of a heterologous mannosidase enzyme encoded by pBB27-2 ( Saccharomyces  MNN10 (s)/ C. elegans  mannosidase IB Δ31) expressed in  P. pastoris.    
         FIGS. 12A-12C  show MALDI-TOF analysis of N-glycans released from a cell free extract of  K. lactis .  FIG. 12A  shows the N-glycans released from wild-type cells, which includes high-mannose type N-glycans.  FIG. 12B  shows the N-glycans released from och1 mnn1 deleted cells, revealing a distinct peak of mass (m/z) at 1908 consistent with its identification as Man 9 GlcNAc 2  [d]. 
         FIG. 12C  shows the N-glycans released from och1 mnn1 deleted cells after in vitro α-1,2-mannosidase digest corresponding to a peak consistent with Man 5 GlcNAc 2 . 
         FIG. 13  is a schematic of the structure of the dolichyl pyrophosphate-linked oligosaccharide. 
         FIG. 4A-14B  are schematics of the generation of GlcNAc 2 Man 3 GlcNAc 2 N-glycans from fungal host cells which are deficient in alg3, alg9, or alg12 activities. 
         FIG. 15  is a schematic of processing reactions required to produce mammalian-type oligosaccharide structures in a fungal host cell with an alg3, och1 genotype. 
         FIGS. 16A-16F  show  S. cerevisiae  Alg3 Sequence Comparisons (Blast) (SEQ ID NOs:9-20, respectively, in order of appearance) 
         FIG. 17  shows  S. cerevisiae  ALG3 (SEQ ID NO:21) and Alg3p (SEQ ID NO:22) Sequences 
         FIG. 18  shows  P. pastoris  ALG3 (SEQ ID NO:23) and Alg3p (SEQ ID NO:24) Sequences 
         FIGS. 19A-19D  show  P. pastoris  ALG3 Sequence Comparisons (Blast) (SEQ ID NOs:23-31, respectively, in order of appearance) 
         FIG. 20  shows  K. lactis  ALG3 (SEQ ID NO:33) and Alg3p (SEQ ID NO:34) Sequences 
         FIG. 21  shows  K. lactis  ALG3 Sequence Comparisons (Blast) (SEQ ID NOs:35-40, respectively, in order of appearance) 
         FIG. 22  shows a model of an IgG immunoglobulin. Heavy chain and light chain can be, based on similar secondary and tertiary structure, subdivided into domains. The two heavy chains (domains V H , C H 1, C H 2 and C H 3) are linked through three disulfide bridges. The light chains (domains V L  and C L ) are linked by another disulfide bridge to the C H 1 portion of the heavy chain and, together with the C H 1 and V H  fragments, make up the Fab region. Antigens bind to the terminal portion of the Fab region. Effector-functions, such as Fc-gamma-Receptor binding have been localized to the C H 2 domain, just downstream of the hinge region and are influenced by N-glycosylation of asparagine 297 in the heavy chain. 
         FIG. 23  is a schematic overview of a modular IgG1 expression vector. 
         FIG. 24  shows  M. musculus  GnTIII Nucleic Acid (SEQ ID NO:45) And Amino Acid (SEQ ID NO:46) Sequences 
         FIG. 25  (top) is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a  P. pastoris  YSH-1 displaying a predominant peak at 1461 m/z corresponding to the mass of GlcNAcMan 5 GlcNAc 2  [d];  FIG. 25  (bottom) shows a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a  P. pastoris  YSH-1 transformed with  D. melanogaster  mannosidase IIΔ74/ S. cerevisiae  MNN2(s) showing a predominant peak at 1140 m/z corresponding to the mass of GlcNAcMan 3 GlcNAc 2  [b] and other peaks corresponding to GlcNAcMan 4 GlcNAc 2  [c] at 1303 m/z and GlcNAcMan 5 GlcNAc 2  [d] at 1465 m/z. This strain was designated YSH-37. 
         FIG. 26  (top) is the MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in  P. pastoris  YSH-1 as shown in  FIG. 25  (top);  FIG. 26  (bottom) is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  YSH-1 cells transformed with a pVA53 construct ( S. cerevisiae  MNN2(s)/mGnTIII). The peak at 1463 m/z corresponds the mass of GlcNAcMan 5 GlcNAc 2  [d] and the peak at 1666 m/z corresponds to the mass of GlcNAc 2 Man 5 GlcNAc 2  [a]. 
         FIG. 27  (top) is the MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in  P. pastoris  YSH-1 as shown in  FIG. 25  (top);  FIG. 27  (bottom) is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  YSH-1 cells transformed with a pVA55 construct ( S. cerevisiae  MNN2(s)/mGnTIII). The peak at 1463 m/z corresponds to the mass of GlcNAcMan 5 GlcNAc 2  [d] and the peak at 1667 m/z corresponds to the mass of GlcNAc 2 Man 5 GlcNAc 2  [a]. 
         FIG. 28  (top) is the MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in  P. pastoris  YSH-1 as shown in  FIG. 25  (top);  FIG. 28  (bottom) is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  YSH-1 cells transformed with a pVB51 construct (K lactic GNT1(s)/mGnTIII). The predominant peak at 1463 m/z corresponds to the mass of GlcNAcMan 5 GlcNAc 2  [d] and a second peak at 1726 m/z [e], which does not correspond to the mass of GlcNAc 2 Man 5 GlcNAc 2  is observed. 
         FIG. 29  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  YSH-44 cells. The predominant peak at 1356 m/z corresponds to the mass of GlcNAc 2 Man 3 GlcNAc 2  [x]. 
         FIG. 30  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  YSH-44 cells transformed with a pVA53 construct ( S. cerevisiae  MNN2(s)/mGnTIII). The peak at 1340 m/z corresponds to the mass of GlcNAc 2 Man 3 GlcNAc 2  [x] and the peak at 1542 m/z corresponds to the mass of GlcNAc 3 Man 3 GlcNAc 2  [y]. 
         FIG. 31  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP6-5 cells. The predominant peak at 1340 m/z corresponds to the mass of GlcNAc 2 Man 3 GlcNAc 2  [x]. 
         FIG. 32  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP6-5 cells transformed with a pVA53 construct ( S. cerevisiae  MNN2(s)/mGnTIII). The peak at 1340 m/z corresponds to the mass of GlcNAc 2 Man 3 GlcNAc 2  [x] and the peak at 1543 m/z corresponds to the mass of GlcNAc 3 Man 3 GlcNAc 2  [y]. 
         FIGS. 33A-33D  show a high performance liquid chromatogram, which demonstrates a lack of extracellular GnTIII activity (pVA53) in the supernatant. The N-glycan GlcNAcMan 5 GlcNAc 2  purified from K3 expressed in PBP-3 strain was added to: BMMY (A); 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) in BMMY (B); the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C); and the supernatant of YSH-57+1 mM UDP-GlcNAc (D). 
         FIG. 34  shows a high performance liquid chromatogram, which demonstrates a lack of extracellular GnTIII activity (pVA53) in the supernatant. The N-glycan GlcNAc 2 Man 3 GlcNAc 2  purified from K3 expressed in YSH-44 strain was added to: BMMY (A); 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) in BMMY (B); and the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C). 
         FIGS. 35A-35B  are schematic diagrams comparing the normal glycosylation pathways in humans and  P. pastoris  (Panel A) with an engineered humanized N-glycosylation pathway in lower eukaryotes (Panel B). The engineered pathway represents the construction of  P. pastoris  strain PBP6-5, which after modification with GnTIII becomes  P. pastoris  strain PBP38. 
         FIG. 36  is a schematic diagram showing the predominant secreted glycoform produced by each of the designated  P. pastoris  strains and the gene modification used to engineer each of the strains. 
         FIG. 37  is a structural representation of the transfer of a GlcNAc to the oligosaccharide intermediate, GlcNAcMan 5 GlcNAc 2 , produced on glycoproteins in a lower eukaryotic host cell, as catalyzed by GnTIII. 
         FIG. 38  is a structural representation of the transfer of a GlcNAc to the oligosaccharide intermediate, GlcNAcMan 3 GlcNAc 2 , produced on glycoproteins in a lower eukaryotic host cell, as catalyzed by GnTII, and the subsequent transfer of a GlcNAc to the product of that reaction, GlcNAc 2 Man 3 GlcNAc 2 , as catalyzed by GnTIII. 
         FIG. 39  is a schematic diagram showing the transfer of a UDP-GlcNAc residue onto oligosaccharide intermediates catalyzed by GnTIV, GnTV, GnTVI and GnTIX in  P. pastoris.    
         FIGS. 40A-40C  show three representative plasmid maps  FIG. 40A : pPB144 containing a gene fragment encoding mouse GnTIV;  FIG. 40B : pPB140 containing a gene fragment encoding human GnTV; and  FIG. 40C : pPB176 containing a gene fragment encoding mouse GnTIX used for transformation in a host  P. pastoris.    
         FIGS. 41A-41B  show the nucleotide sequence (SEQ ID NO:123) of the gene encoding the  Homo sapiens  mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isoenzyme A (MGAT4A) (SEQ ID NO:122) Accession number NM — 012214. 
         FIGS. 42A-42B  show the nucleotide sequence (SEQ ID NO:40) of the gene encoding the  Homo sapiens  mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isoenzyme B (MGAT4B) (SEQ ID NO:122) Accession number NM — 014275. 
         FIGS. 43A-43B  show the nucleotide sequence (SEQ ID NO:44) of the gene encoding the  Mus musculus  N-acetylglucosaminyltransferase V (Mgat5) (SEQ ID NO:43) Accession number AF474154. 
         FIGS. 44A-44B  show shows the nucleotide sequence (SEQ ID NO:51) of the gene encoding the  Gallus gallus  N-acetylglucosaminyltransferase VI (SEQ ID NO:50) Accession number AB040608. 
         FIGS. 45A-45B  show the nucleotide sequence (SEQ ID NO:120) of the gene encoding the  Homo sapiens  N-acetylglucosaminyltransferase IX (SEQ ID NO:119) Accession number AB 109185.1. 
         FIGS. 46A-46B  show the codon optimized DNA fragment encoding part of the human N-acetylglucosaminyltransferase IX lacking the TM domain (Δ43) (SEQ ID NO:121). 
         FIG. 47  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP43. The yeast strain  P. pastoris  YSH-44 was transformed with pPB144 containing the  S. cerevisiae  MNN2(s)/human GnTIV fusion construct. The peak at 1543 m/z corresponds to the mass of GlcNAc 3 Man 3 GlcNAc 2  [y]. 
         FIG. 48  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP32. The yeast strain  P. pastoris  YSH-44 was transformed with pPB140 containing the  S. cerevisiae  MNN2(s)/mouse GnTV fusion. The peak at 1559 m/z corresponds to the mass of GlcNAc 3 Man 3 GlcNAc 2  [y]. 
         FIG. 49  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP46. The yeast strain  P. pastoris  YSH-44 was transformed with pPB140 and pPB144. The peak at 1543 m/z corresponds to the mass of GlcNAc 3 Man 3 GlcNAc 2  [y] and the peak at 1747 m/z corresponds to the mass of GlcNAc 4 Man 3 GlcNAc 2  [z]. 
         FIG. 50  is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein expressed in  P. pastoris  PBP94. The yeast strain  P. pastoris  YSH-44 was transformed with pPB128 containing the  S. cerevisiae  MNN2(s)/mouse GnTIVA fusion construct and pPB140 containing the  S. cerevisiae  MNN2(s)/mouse GnTV fusion construct. The predominant peak at 1743 m/z corresponds to the mass of GlcNAc 4 Man 3 GlcNAc 2  [z]. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. 
     The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,  Current Protocols in Molecular Biology , Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane,  Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990);  Introduction to Glycobiology , Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;  Handbook of Biochemistry: Section A Proteins , Vol I 1976 CRC Press;  Handbook of Biochemistry: Section A Proteins , Vol II 1976 CRC Press;  Essentials of Glycobiology , Cold Spring Harbor Laboratory Press (1999). The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. 
     All publications, patents and other references mentioned herein are incorporated by reference. 
     The following terms, unless otherwise indicated, shall be understood to have the following meanings: 
     As used herein, the term “N-glycan” refers to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-glycans have a common pentasaccharide core of Man 3 GlcNAc 2  (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannose core” used with respect to the N-glycan also refers to the structure Man 3 GlcNAc 2  (“Man 3 ”). The term “pentamannose core” or “Mannose-5 core” or “Man 5 ” used with respect to the N-glycan refers to the structure Man 5 GlcNAc 2 . N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, fucose, and sialic acid) that are attached to the Man 3  core structure. N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid). 
     A “high mannose” type N-glycan has five or more mannose residues. A “complex” type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of the trimannose core. Complex N-glycans may also have galactose (“Gal”) residues that are optionally modified with sialic acid or derivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl). A complex N-glycan typically has at least one branch that terminates in an oligosaccharide such as, for example: NeuNAc-; NeuAcα2-6GalNAcα1-; NeuAcα2-3Galβ1-3GalNAcα1-; NeuAcα2-3/6Galβ1-4GlcNAcβ1-; GlcNAcα1-4Galβ1-(mucins only); Fucα1-2Galβ1-(blood group H). Sulfate esters can occur on galactose, GalNAc, and GlcNAc residues, and phosphate esters can occur on mannose residues. NeuAc (Neu: neuraminic acid; Ac: acetyl) can be O-acetylated or replaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core. 
     The term “predominant” or “predominantly” used with respect to the production of N-glycans refers to a structure which represents the major peak detected by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analysis. 
     Abbreviations used herein are of common usage in the art, see, e.g., abbreviations of sugars, above. Other common abbreviations include “PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAc Tr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes; “NANA” refers to N-acetylneuraminic acid. 
     As used herein, a “humanized glycoprotein” or a “human-like glycoprotein” refers alternatively to a protein having attached thereto N-glycans having fewer than four mannose residues, and synthetic glycoprotein intermediates (which are also useful and can be manipulated further in vitro or in vivo) having at least five mannose residues. Preferably, glycoproteins produced according to the invention contain at least 30 mole %, preferably at least 40 mole % and more preferably 50, 60, 70, 80, 90, or even 100 mole % of the Man 5 GlcNAc 2  intermediate, at least transiently. This may be achieved, e.g., by engineering a host cell of the invention to express a “better”, i.e., a more efficient glycosylation enzyme. For example, a mannosidase is selected such that it will have optimal activity under the conditions present at the site in the host cell where proteins are glycosylated and is introduced into the host cell preferably by targeting the enzyme to a host cell organelle where activity is desired. 
     The term “enzyme”, when used herein in connection with altering host cell glycosylation, refers to a molecule having at least one enzymatic activity, and includes full-length enzymes, catalytically active fragments, chimerics, complexes, and the like. A “catalytically active fragment” of an enzyme refers to a polypeptide having a detectable level of functional (enzymatic) activity. Enzyme activity is “substantially intracellular” when less than 10% of the enzyme activity is measurable outside the cell compared to that measurable from lysed cells. 
     A lower eukaryotic host cell, when used herein in connection with glycosylation profiles, refers to most typical lower eukaryotic cells, including uni- and multicellular fungal and algal cells. 
     As used herein, the term “secretion pathway” refers to the assembly line of various glycosylation enzymes to which a lipid-linked oligosaccharide precursor and an N-glycan substrate are sequentially exposed, following the molecular flow of a nascent polypeptide chain from the cytoplasm to the endoplasmic reticulum (ER) and the compartments of the Golgi apparatus. Enzymes are said to be localized along this pathway. An enzyme X that acts on a lipid-linked glycan or an N-glycan before enzyme Y is said to be or to act “upstream” to enzyme Y; similarly, enzyme Y is or acts “downstream” from enzyme X. 
     The term “targeting peptide” as used herein refers to nucleotide or amino acid sequences encoding a cellular targeting signal peptide which mediates the localization (or retention) of an associated sequence to sub-cellular locations, e.g., organelles. 
     The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. 
     The term includes single and double stranded forms of DNA. A nucleic acid molecule of this invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. 
     Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X” refers to a nucleic acid, at least a portion of which has either (I) the sequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ ID NO:X. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. 
     An “isolated” or “substantially pure” nucleic acid or polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases, and genomic sequences with which it is naturally associated. The term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “isolated” or “substantially pure” also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems. 
     However, “isolated” does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed “isolated” herein if a heterologous sequence (i.e., a sequence that is not naturally adjacent to this endogenous nucleic acid sequence) is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. By way of example, a non-native promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a human cell, such that this gene has an altered expression pattern. This gene would now become “isolated” because it is separated from at least some of the sequences that naturally flank it. 
     A nucleic acid is also considered “isolated” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “isolated” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. An “isolated nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, a nucleic acid construct present as an episome. Moreover, an “isolated nucleic acid” can be substantially free of other cellular material, or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. 
     As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. 
     The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990)  Methods Enzymol.  183:63-98, incorporated herein by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. 
     The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. 
     Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. 
     In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the T m  for the specific DNA hybrid under a particular set of conditions. The T m  is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., supra, page 9.51, hereby incorporated by reference. For purposes herein, “high stringency conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled artisan that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing. 
     The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. See, e.g., Leung et al. (1989)  Technique  1:11-15 and Caldwell and Joyce (1992)  PCR Methods Applic.  2:28-33); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest. See, e.g., Reidhaar-Olson et al. (1988)  Science  241:53-57). 
     The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). 
     “Operatively linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest. 
     The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. 
     The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a nucleic acid such as a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. 
     The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function. 
     The term “polypeptide” as used herein encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. 
     The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) when it exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well-known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment. 
     The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long. 
     A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well-known in the art, and include radioactive isotopes such as  125 I,  32 P,  35 S, and  3 H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well-known in the art. See Ausubel et al.,  Current Protocols in Molecular Biology , Greene Publishing Associates (1992, and Supplements to 2002), hereby incorporated by reference. 
     A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. 
     A mutein has at least 70% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having 80%, 85% or 90% overall sequence homology to the wild-type protein. In an even more preferred embodiment, a mutein exhibits 95% sequence identity, even more preferably 97%, even more preferably 98% and even more preferably 99% overall sequence identity. Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit. 
     Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs. 
     As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See  Immunology—A Synthesis  (2 nd  Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. 
     A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences). In a preferred embodiment, a homologous protein is one that exhibits 60% sequence homology to the wild type protein, more preferred is 70% sequence homology. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence homology to the wild type protein. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence identity. As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function. 
     When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson (1990)  Methods Enzymol.  183:63-98). 
     The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). 
     Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. 
     A preferred algorithm when comparing a inhibitory molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al. (1990)  J. Mol. Biol.  215:403-410; Gish and States (1993)  Nature Genet.  3:266-272; Madden et al. (1996)  Meth. Enzymol.  266:131-141; Altschul et al. (1997)  Nucleic Acids Res.  25:3389-3402; Zhang and Madden (1997)  Genome Res.  7:649-656), especially blastp or tblastn (Altschul et al., 1997). Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. 
     The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see Pearson (1990)  Methods Enzymol.  183:63-98). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference. 
     The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in-frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. 
     The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. 
     The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. 
     As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. 
     Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 
     Methods for Producing Human-Like Glycoproteins in Lower Eukaryotic Host Cells 
     The invention provides methods for producing a glycoprotein having human-like glycosylation in a non-human eukaryotic host cell. As described in more detail below, a eukaryotic host cell that does not naturally express, or which is engineered not to express, one or more enzymes involved in production of high mannose structures is selected as a starting host cell. Such a selected host cell is engineered to express one or more enzymes or other factors required to produce human-like glycoproteins. A desired host strain can be engineered one enzyme or more than one enzyme at a time. In addition, a nucleic acid molecule encoding one or more enzymes or activities may be used to engineer a host strain of the invention. Preferably, a library of nucleic acid molecules encoding potentially useful enzymes (e.g., chimeric enzymes comprising a catalytically active enzyme fragment ligated in-frame to a heterologous subcellular targeting sequence) is created (e.g., by ligation of sub-libraries comprising enzymatic fragments and subcellular targeting sequences), and a strain having one or more enzymes with optimal activities or producing the most “human-like” glycoproteins may be selected by transforming target host cells with one or more members of the library. 
     In particular, the methods described herein enable one to obtain, in vivo, Man 5 GlcNAc 2  structures in high yield, at least transiently, for the purpose of further modifying it to yield complex N-glycans. A successful scheme to obtain suitable Man 5 GlcNAc 2  structures in appropriate yields in a host cell, such as a lower eukaryotic organism, generally involves two parallel approaches: (1) reducing high mannose structures made by endogenous mannosyltransferase activities, if any, and (2) removing 1,2-α-mannose by mannosidases to yield high levels of suitable Man 5 GlcNAc 2  structures which may be further reacted inside the host cell to form complex, human-like glycoforms. 
     Accordingly, a first step involves the selection or creation of a eukaryotic host cell, e.g., a lower eukaryote, capable of producing a specific precursor structure of Man 5 GlcNAc 2  that is able to accept in vivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In one embodiment, the method involves making or using a non-human eukaryotic host cell depleted in a 1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein. Preferably, the host cell is depleted in an initiating 1,6 mannosyltransferase activity (see below). Such a host cell will lack one or more enzymes involved in the production of high mannose structures which are undesirable for producing human-like glycoproteins. 
     One or more enzyme activities are then introduced into such a host cell to produce N-glycans within the host cell characterized by having at least 30 mol % of the Man 5 GlcNAc 2  (“Man 5 ”) carbohydrate structures. Man 5 GlcNAc 2  structures are necessary for complex N-glycan formation: Man 5 GlcNAc 2  must be formed in vivo in a high yield (e.g., in excess of 30%), at least transiently, as subsequent mammalian- and human-like glycosylation reactions require Man 5 GlcNAc 2  or a derivative thereof. 
     This step also requires the formation of a particular isomeric structure of Man 5 GlcNAc 2  within the cell at a high yield. While Man 5 GlcNAc 2  structures are necessary for complex N-glycan formation, their presence is by no means sufficient. That is because Man 5 GlcNAc 2  may occur in different isomeric forms, which may or may not serve as a substrate for GlcNAc transferase I. As most glycosylation reactions are not complete, a particular glycosylated protein generally contains a range of different carbohydrate structures (i.e., glycoforms) on its surface. Thus, the mere presence of trace amounts (i.e., less than 5%) of a particular structure like Man 5 GlcNAc 2  is of little practical relevance for producing mammalian- or human-like glycoproteins. It is the formation of a GlcNAc transferase I-accepting Man 5 GlcNAc 2  intermediate ( FIG. 1B ) in high yield (i.e., above 30%), which is required. The formation of this intermediate is necessary to enable subsequent in vivo synthesis of complex N-glycans on glycosylated proteins of interest (target proteins). 
     Accordingly, some or all of the Man 5 GlcNAc 2  produced by the selected host cell must be a productive substrate for enzyme activities along a mammalian glycosylation pathway, e.g., can serve as a substrate for a GlcNAc transferase I activity in vivo, thereby forming the human-like N-glycan intermediate GlcNAcMan 5 GlcNAc 2  in the host cell. In a preferred embodiment, at least 10%, more preferably at least 30% and most preferably 50% or more of the Man 5 GlcNAc 2  intermediate produced in the host cell of the invention is a productive substrate for GnTI in vivo. It is understood that if, for example, GlcNAcMan 5 GlcNAc 2  is produced at 10% and Man 5 GlcNAc 2  is produced at 25% on a target protein, that the total amount of transiently produced Man 5 GlcNAc 2  is 35% because GlcNAcMan 5 GlcNAc 2  is a product of Man 5 GlcNAc 2 . 
     One of ordinary skill in the art can select host cells from nature, e.g., existing fungi or other lower eukaryotes that produce significant levels of Man 5 GlcNAc 2  in vivo. As yet, however, no lower eukaryote has been shown to provide such structures in vivo in excess of 1.8% of the total N-glycans (see e.g. Maras et al. (1997)  Eur. J. Biochem.  249:701-707). Alternatively, such host cells may be genetically engineered to produce the Man 5 GlcNAc 2  structure in vivo. Methods such as those described in U.S. Pat. No. 5,595,900 may be used to identify the absence or presence of particular glycosyltransferases, mannosidases and sugar nucleotide transporters in a target host cell or organism of interest. 
     Inactivation of Undesirable Host Cell Glycosylation Enzymes 
     The methods of the invention are directed to making host cells which produce glycoproteins having altered, and preferably human-like, N-glycan structures. In a preferred embodiment, the methods are directed to making host cells in which oligosaccharide precursors are enriched in Man 5 GlcNAc 2 . Preferably, a eukaryotic host cell is used that does not express one or more enzymes involved in the production of high mannose structures. Such a host cell may be found in nature or may be engineered, e.g., starting with or derived from one of many such mutants already described in yeasts. Thus, depending on the selected host cell, one or a number of genes that encode enzymes known to be characteristic of non-human glycosylation reactions will have to be deleted. Such genes and their corresponding proteins have been extensively characterized in a number of lower eukaryotes (e.g.,  S. cerevisiae, T. reesei, A. nidulans , etc.), thereby providing a list of known glycosyltransferases in lower eukaryotes, their activities and their respective genetic sequence. These genes are likely to be selected from the group of mannosyltransferases, e.g. 1,3 mannosyltransferases (e.g. MNN1 in  S. cerevisiae ) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KRE family from  S. cerevisiae ), 1,6 mannosyltransferases (OCH1 from  S. cerevisiae ), mannosylphosphate transferases and their regulators (MNN4 and MNN6 from  S. cerevisiae ) and additional enzymes that are involved in aberrant, i.e., non-human, glycosylation reactions. Many of these genes have in fact been deleted individually giving rise to viable phenotypes with altered glycosylation profiles. Examples are shown in Table 1. 
     Preferred lower eukaryotic host cells of the invention, as described herein to exemplify the required manipulation steps, are hypermannosylation-minus (och1) mutants of  Pichia pastoris  or  K. lactis . Like other lower eukaryotes,  P. pastoris  processes Man 9 GlcNAc 2  structures in the ER with an α-1,2-mannosidase to yield Man 8 GlcNAc 2  ( FIG. 1A ). Through the action of several mannosyltransferases, this structure is then converted to hypermannosylated structures (Man &gt;9 GlcNAc 2 ), also known as mannans ( FIG. 35A ). In addition, it has been found that  P. pastoris  is able to add non-terminal phosphate groups, through the action of mannosylphosphate transferases, to the carbohydrate structure. This differs from the reactions performed in mammalian cells, which involve the removal rather than addition of mannose sugars ( FIG. 35A ). It is of particular importance to eliminate the ability of the eukaryotic host cell, e.g., fungus, to hypermannosylate an existing Man 8 GlcNAc 2  structure. This can be achieved by either selecting for a host cell that does not hypermannosylate or by genetically engineering such a cell. 
     Genes that are involved in the hypermannosylation process have been identified, e.g., in  Pichia pastoris , and by creating mutations in these genes, one can reduce the production of “undesirable” glycoforms. Such genes can be identified by homology to existing mannosyltransferases or their regulators (e.g., OCH1, MNN4, MNN6, MNN1) found in other lower eukaryotes such as  C. albicans, Pichia angusta  or  S. cerevisiae  or by mutagenizing the host strain and selecting for a glycosylation phenotype with reduced mannosylation. Based on homologies amongst known mannosyltransferases and mannosylphosphate transferases, one may either design PCR primers (examples of which are shown in Table 2), or use genes or gene fragments encoding such enzymes as probes to identify homologs in DNA libraries of the target or a related organism. Alternatively, one may identify a functional homolog having mannosyltransferase activity by its ability to complement particular glycosylation phenotypes in related organisms. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 PCR Primers 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Target Gene(s) in  P.   
                   
               
               
                 PCR primer A 
                 PCR primer B 
                 
                   pastoris 
                 
                 Homologs 
               
               
                   
               
               
                 ATGGCGAAGGCA 
                 TTAGTCCTTCCAA 
                 1,6- 
                 OCH1  S. cerevisiae , 
               
               
                 GATGGCAGT 
                 CTTCCTTC 
                 mannosyltransferase 
                 
                   Pichia albicans 
                 
               
               
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 4) 
                   
                   
               
               
                   
               
               
                 TAYTGGMGNGTN 
                 GCRTCNCCCCANC 
                 1,2 
                 KTR/KRE family,  S.   
               
               
                 GARCYNGAYATH 
                 KYTCRTA 
                 mannosyltransferases 
                 
                   cerevisiae 
                 
               
               
                 AA 
                 (SEQ ID NO: 6) 
                   
                   
               
               
                 (SEQ ID NO: 5) 
               
               
                   
               
               
                 Legend: 
               
               
                 M = A or C, R = A or G, W = A or T, S = C or G, Y = C or T, K = G or T, V = A or C or G, H = A or C or T, D = A or G or T, B = C or G or T, N = G or A or T or C. 
               
            
           
         
       
     
     To obtain the gene or genes encoding 1,6-mannosyltransferase activity in  P. pastoris , for example, one would carry out the following steps: OCH1 mutants of  S. cerevisiae  are temperature sensitive and are slow growers at elevated temperatures. One can thus identify functional homologs of OCH1 in  P. pastoris  by complementing an OCH1 mutant of  S. cerevisiae  with a  P. pastoris  DNA or cDNA library. Mutants of  S. cerevisiae  are available, e.g., from Stanford University, and are commercially available from ResGen, Invitrogen Corp. (Carlsbad, Calif.). Mutants that display a normal growth phenotype at elevated temperature, after having been transformed with a  P. pastoris  DNA library, are likely to carry an OCH1 homolog of  P. pastoris . Such a library can be created by partially digesting chromosomal DNA of  P. pastoris  with a suitable restriction enzyme and, after inactivating the restriction enzyme, ligating the digested DNA into a suitable vector, which has been digested with a compatible restriction enzyme. 
     Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4) plasmid based on pBluescript containing the Trp1 marker (Sikorski and Hieter (1989)  Genetics  122:19-27) and pFL44S, a high copy (2μ) plasmid based on a modified pUC19 containing the URA3 marker (Bonneaud et al. (1991)  Yeast  7:609-615). Such vectors are commonly used by academic researchers and similar vectors are available from a number of different vendors (e.g., Invitrogen (Carlsbad, Calif.); Pharmacia (Piscataway, N.J.); New England Biolabs (Beverly, Mass.)). Further examples include pYES/GS, 2μ origin of replication based yeast expression plasmid from Invitrogen, or Yep24 cloning vehicle from New England Biolabs. 
     After ligation of the chromosomal DNA and the vector, one may transform the DNA library into a strain of  S. cerevisiae  with a specific mutation and select for the correction of the corresponding phenotype. After sub-cloning and sequencing the DNA fragment that is able to restore the wild-type phenotype, one may use this fragment to eliminate the activity of the gene product encoded by OCH1 in  P. pastoris  using in vivo mutagenesis and/or recombination techniques well-known to those skilled in the art. 
     Alternatively, if the entire genomic sequence of a particular host cell, e.g., fungus, of interest is known, one may identify such genes simply by searching publicly available DNA databases, which are available from several sources, such as NCBI, Swissprot. For example, by searching a given genomic sequence or database with sequences from a known 1,6 mannosyltransferase gene (e.g., OCH1 from  S. cerevisiae ), one can identify genes of high homology in such a host cell genome which may (but do not necessarily) encode proteins that have 1,6-mannosyltransferase activity. Nucleic acid sequence homology alone is not enough to prove, however, that one has identified and isolated a homolog encoding an enzyme having the same activity. To date, for example, no data exist to show that an OCH1 deletion in  P. pastoris  eliminates the crucial initiating 1,6-mannosyltransferase activity (Martinet et al. (1998) Biotech. Letters 20(12):1171-1177; Contreras et al. WO 02/00856 A2). Thus, no data prove that the  P. pastoris  OCH1 gene homolog actually encodes that function. That demonstration is provided for the first time herein. 
     Homologs to several  S. cerevisiae  mannosyltransferases have been identified in  P. pastoris  using these approaches. Homologous genes often have similar functions to genes involved in the mannosylation of proteins in  S. cerevisiae  and thus their deletion may be used to manipulate the glycosylation pattern in  P. pastoris  or, by analogy, in any other host cell, e.g., fungus, plant, insect or animal cells, with similar glycosylation pathways. 
     The creation of gene knock-outs, once a given target gene sequence has been determined, is a well-established technique in the art and can be carried out by one of ordinary skill in the art (see, e.g., Rothstein (1991)  Methods in Enzymology  194:281). The choice of a host organism may be influenced by the availability of good transformation and gene disruption techniques. 
     If several mannosyltransferases are to be knocked out, the method developed by Alani and Kleckner (1987)  Genetics  116:541-545, for example, enables the repeated use of a selectable marker, e.g., the URA3 marker in yeast, to sequentially eliminate all undesirable endogenous mannosyltransferase activity. This technique has been refined by others but basically involves the use of two repeated DNA sequences, flanking a counter selectable marker. For example: URA3 may be used as a marker to ensure the selection of a transformants that have integrated a construct. By flanking the URA3 marker with direct repeats one may first select for transformants that have integrated the construct and have thus disrupted the target gene. After isolation of the transformants, and their characterization, one may counter select in a second round for those that are resistant to 5-fluoroorotic acid (5-FOA). Colonies that are able to survive on plates containing 5-FOA have lost the URA3 marker again through a crossover event involving the repeats mentioned earlier. This approach thus allows for the repeated use of the same marker and facilitates the disruption of multiple genes without requiring additional markers. Similar techniques for sequential elimination of genes adapted for use in another eukaryotic host cells with other selectable and counter-selectable markers may also be used. 
     Eliminating specific mannosyltransferases, such as 1,6 mannosyltransferase (OCH1) or mannosyiphosphate transferases (MNN6, or genes complementing lbd mutants) or regulators (MNN4) in  P. pastoris  enables one to create engineered strains of this organism which synthesize primarily Man 8 GlcNAc 2  and which can be used to further modify the glycosylation pattern to resemble more complex glycoform structures, e.g., those produced in mammalian, e.g., human cells. A preferred embodiment of this method utilizes DNA sequences encoding biochemical glycosylation activities to eliminate similar or identical biochemical functions in  P. pastoris  to modify the glycosylation structure of glycoproteins produced in the genetically altered  P. pastoris  strain. 
     Methods used to engineer the glycosylation pathway in yeasts as exemplified herein can be used in filamentous fungi to produce a preferred substrate for subsequent modification. Strategies for modifying glycosylation pathways in  A. niger  and other filamentous fungi, for example, can be developed using protocols analogous to those described herein for engineering strains to produce human-like glycoproteins in yeast. Undesired gene activities involved in 1,2 mannosyltransferase activity, e.g., KTR/KRE homologs, are modified or eliminated. A filamentous fungus, such as  Aspergillus , is a preferred host because it lacks the 1,6 mannosyltransferase activity and as such, one would not expect a hypermannosylating gene activity, e.g. OCH1, in this host. By contrast, other desired activities (e.g., α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT), galactosyltransferase (GalT) and sialyltransferase (ST)) involved in glycosylation are introduced into the host using the targeting methods of the invention. 
     Engineering or Selecting Hosts Having Diminished Initiating α-1,6 Mannosyltransferase Activity 
     In a preferred embodiment, the method of the invention involves making or using a host cell which is diminished or depleted in the activity of an initiating α-1,6-mannosyltransferase, i.e., an initiation specific enzyme that initiates outer chain mannosylation on the α-1,3 arm of the Man 3 GlcNAc 2  core structure. In  S. cerevisiae , this enzyme is encoded by the OCH1 gene. Disruption of the OCH1 gene in  S. cerevisiae  results in a phenotype in which N-linked sugars completely lack the poly-mannose outer chain. Previous approaches for obtaining mammalian-type glycosylation in fungal strains have required inactivation of OCH1 (see, e.g., Chiba et al. (1998)  J. Biol. Chem.  273:26298-304). Disruption of the initiating α-1,6-mannosyltransferase activity in a host cell of the invention may be optional, however (depending on the selected host cell), as the Och1p enzyme requires an intact Man 8 GlcNAc 2  for efficient mannose outer chain initiation. Thus, host cells selected or produced according to this invention which accumulate oligosaccharides having seven or fewer mannose residues may produce hypoglycosylated N-glycans that will likely be poor substrates for Och1p (see, e.g., Nakayama et al. (1997)  FEBS Lett.  412(3):547-50). 
     The OCH1 gene was cloned from  P. pastoris  (Example 1) and  K. lactis  (Example 9), as described. The nucleic acid and amino acid sequences of the OCH1 gene from  K. lactis  are set forth in SEQ ID NOs:7 and 8. Using gene-specific primers, a construct was made from each clone to delete the OCH1 gene from the genome of  P. pastoris  and  K. lactis  (Examples 1 and 9, respectively). Host cells depleted in initiating α-1,6-mannosyltransferase activity and engineered to produce N-glycans having a Man 5 GlcNAc 2  carbohydrate structure were thereby obtained (see, e.g.,  FIGS. 5 ,  6 , and  12 ; Examples 4 and 9). 
     Thus, in another embodiment, the invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of at least forty-five, preferably at least 50, more preferably at least 60 and most preferably 75 or more nucleotide residues of the  K. lactis  OCH1 gene (SEQ ID NO: 7), and homologs, variants and derivatives thereof. The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. Similarly, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided. Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the invention, as described further herein. Similarly, host cells transformed with the nucleic acid molecules or vectors of the invention are provided. 
     The invention further provides methods of making or using a non-human eukaryotic host cell diminished or depleted in an alg gene activity (i.e., alg activities, including equivalent enzymatic activities in non-fungal host cells) and introducing into the host cell at least one glycosidase activity. In a preferred embodiment, the glycosidase activity is introduced by causing expression of one or more mannosidase activities within the host cell, for example, by activation of a mannosidase activity, or by expression from a nucleic acid molecule of a mannosidase activity, in the host cell. 
     In another embodiment, the method involves making or using a host cell diminished or depleted in the activity of one or more enzymes that transfer a sugar residue to the 1,6 arm of lipid-linked oligosaccharide precursors ( FIG. 13 ). A host cell of the invention is selected for or is engineered by introducing a mutation in one or more of the genes encoding an enzyme that transfers a sugar residue (e.g., mannosylates) the 1,6 arm of a lipid-linked oligosaccharide precursor. The sugar residue is more preferably mannose, is preferably a glucose, GlcNAc, galactose, sialic acid, fucose or GlcNAc phosphate residue. In a preferred embodiment, the activity of one or more enzymes that mannosylate the 1,6 arm of lipid-linked oligosaccharide precursors is diminished or depleted. The method may further comprise the step of introducing into the host cell at least one glycosidase activity (see below). 
     In yet another embodiment, the invention provides a method for producing a human-like glycoprotein in a non-human host, wherein the glycoprotein comprises an N-glycan having at least two GlcNAcs attached to a trimannose core structure. 
     In each above embodiment, the method is directed to making a host cell in which the lipid-linked oligosaccharide precursors are enriched in Man x GlcNAc 2  structures, where X is 3, 4 or 5 ( FIG. 14 ). These structures are transferred in the ER of the host cell onto nascent polypeptide chains by an oligosaccharyl-transferase and may then be processed by treatment with glycosidases (e.g., α-mannosidases) and glycosyltransferases (e.g., GnTI) to produce N-glycans having GlcNAcManxGlcNAc 2  core structures, wherein X is 3, 4 or 5, and is preferably 3 ( FIGS. 14 and 15 ). As shown in  FIG. 14 , N-glycans having a GlcNAcMan x GlcNAc 2  core structure where X is greater than 3 may be converted to GlcNAcMan 3 GlcNAc 2 , e.g., by treatment with an α-1,3 and/or α-1,2-1,3 mannosidase activity, where applicable. 
     Additional processing of GlcNAcMan 3 GlcNAc 2  by treatment with glycosyltransferases (e.g., GnTII) produces GlcNAc 2 Man 3 GlcNAc 2  core structures which may then be modified, as desired, e.g., by ex vivo treatment or by heterologous expression in the host cell of a set of glycosylation enzymes, including glycosyltransferases, sugar transporters and mannosidases (see below), to become human-like N-glycans. Preferred human-like glycoproteins which may be produced according to the invention include those which comprise N-glycans having seven or fewer, or three or fewer, mannose residues; comprise one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose; and comprise at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man. 
     In one embodiment, the host cell has diminished or depleted Dol-P-Man:Man 5 GlcNAc 2 -PP-Dol Mannosyltransferase activity, which is an activity involved in the first mannosylation step from Man 5 GlcNAc 2 -PP-Dol to Man 6 GlcNAc 2 -PP-Dol at the luminal side of the ER (e.g., ALG3  FIG. 13 ;  FIG. 14 ). In  S. cerevisiae , this enzyme is encoded by the ALG3 gene. As described above,  S. cerevisiae  cells harboring a leaky alg3-1 mutation accumulate Man 5 GlcNAc 2 -PP-Dol and cells having a deletion in alg3 appear to transfer Man 5 GlcNAc 2  structures onto nascent polypeptide chains within the ER. Accordingly, in this embodiment, host cells will accumulate N-glycans enriched in Man 5 GlcNAc 2  structures which can then be converted to GlcNAc 2 Man 3 GlcNAc 2  by treatment with glycosidases (e.g., with α-1,2 mannosidase, α-1,3 mannosidase, or α-1,2-1,3 mannosidase activities) and glycosyltransferase activities (e.g., GnTI, GnTII) ( FIG. 14 ;  FIG. 35B ). 
     As described in Example 10, degenerate primers were designed based on an alignment of Alg3 protein sequences from  S. cerevisiae, D. melanogaster  and humans ( H. sapiens ) ( FIGS. 16 and 17 ), and were used to amplify a product from  P. pastoris  genomic DNA. The resulting PCR product was used as a probe to identify and isolate a  P. pastoris  genomic clone comprising an open reading frame (ORF) that encodes a protein having 35% overall sequence identity and 53% sequence similarity to the  S. cerevisiae  ALG3 gene ( FIGS. 18 and 19 ). This  P. pastoris  gene is referred to herein as “PpALG3”. The ALG3 gene was similarly identified and isolated from  K. lactis  (Example 10;  FIGS. 20 and 21 ). 
     Thus, in another embodiment, the invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of at least forty-five, preferably at least 50, more preferably at least 60 and most preferably 75 or more nucleotide residues of the  P. pastoris  ALG3 gene ( FIG. 18 ) and the  K. lactis  ALG3 gene ( FIG. 20 ), and homologs, variants and derivatives thereof. The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. Similarly, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided ( P. pastoris  and  K. lactis  ALG3 gene products are shown in  FIGS. 18 and 20 ). In addition, also provided are vectors, including expression vectors, which comprise a nucleic acid molecule of the invention, as described further herein. 
     Using gene-specific primers, a construct was made to delete the PpALG3 gene from the genome of  P. pastoris  (Example 10). This strain was used to generate a host cell depleted in Dol-P-Man:Man 5 GlcNAc 2 -PP-Dol Mannosyltransferase activity and produce lipid-linked Man 5 GlcNAc 2 -PP-Dol precursors which are transferred onto nascent polypeptide chains to produce N-glycans having a Man 5 GlcNAc 2  carbohydrate structure. 
     As described in Example 11, such a host cell may be engineered by expression of appropriate mannosidases to produce N-glycans having the desired Man 3 GlcNAc 2  core carbohydrate structure. Expression of GnTs in the host cell (e.g., by targeting a nucleic acid molecule or a library of nucleic acid molecules as described below) enables the modified host cell to produce N-glycans having one or two GlcNAc structures attached to each arm of the Man3 core structure (i.e., GlcNAc 1 Man 3 GlcNAc 2 , GlcNAc 2 Man 3 GlcNAc 2 , or GlcNAc 3 Man 3 GlcNAc 2 ; see  FIG. 15 ). These structures may be processed further using the methods of the invention to produce human-like N-glycans on proteins which enter the secretion pathway of the host cell. 
     In a preferred embodiment, the method of the invention involves making or using a host cell which is both (a) diminished or depleted in the activity of an alg gene or in one or more activities that mannosylate N-glycans on the α-1,6 arm of the Man 3 GlcNAc 2  (“Man3”) core carbohydrate structure; and (b) diminished or depleted in the activity of an initiating α-1,6-mannosyltransferase, i.e., an initiation specific enzyme that initiates outer chain mannosylation (on the α-1,3 arm of the Man3 core structure). In  S. cerevisiae , this enzyme is encoded by the OCH1 gene. Disruption of the och1 gene in  S. cerevisiae  results in a phenotype in which N-linked sugars completely lack the poly-mannose outer chain. Previous approaches for obtaining mammalian-type glycosylation in fungal strains have required inactivation of OCH1 (see, e.g., Chiba et al. (1998)  J. Biol. Chem.  273:26298-304). Disruption of the initiating α-1,6-mannosyltransferase activity in a host cell of the invention is optional, however (depending on the selected host cell), as the Och1p enzyme requires an intact Man 8 GlcNAc for efficient mannose outer chain initiation. Thus, the host cells selected or produced according to this invention, which accumulate lipid-linked oligosaccharides having seven or fewer mannose residues will, after transfer, produce hypoglycosylated N-glycans that will likely be poor substrates for Och1p (see, e.g., Nakayama et al. (1997)  FEBS Lett.  412(3):547-50). 
     Engineering or Selecting Hosts Having N-Acetylglucosaminyltransferase III Activity 
     The invention additionally provides a method for producing a human-like glycoprotein in a lower eukaryotic host cell by expressing an N-acetylglucosaminyltransferase III activity (including a full-length enzyme, homologs, variants, derivatives, and catalytically active fragments thereof). In one embodiment, a host cell (e.g.,  P. pastoris ) is engineered to produce more human-like N-glycans, e.g., by activation of an N-acetylglucosaminyltransferase III activity or by expression from a nucleic acid molecule of an N-acetylglucosaminyltransferase III activity. Using well-known techniques in the art, gene-specific primers are designed to complement the homologous regions of a GnTIII gene, preferably a mammalian GnTIII gene (e.g., mouse GnTIII) ( FIG. 24 ), sequences for which are readily available in the art (e.g., Genbank Accession No. L39373) and are PCR amplified. 
     In one embodiment, the invention provides a method for producing a human-like glycoprotein in a lower eukaryote (e.g.,  P. pastoris ), wherein the glycoprotein comprises an N-glycan exhibiting a bisecting GlcNAc on a trimannose or trimannosyl (Man 3 GlcNAc 2 ) core structure. In this embodiment, GlcNAcMan 3 GlcNAc 2  (which may be produced by reacting a trimannose core with N-acetylglucosaminyltransferase I (“GnTI”) activity, but which is typically produced by trimming of GlcNAcMan 5 GlcNAc 2  by an α-1,3/α-1,6-mannosidase activity, such as Mannosidase II (Hamilton et al. (2003)  Science  301:1244-46)) is reacted with an N-acetylglucosaminyltransferase III activity to produce a bisected GlcNAc 2 Man 3 GlcNAc 2 . Accordingly, the invention provides GnTIII activity, which transfers β-1,4 GlcNAc onto substrates that are capable of accepting the bisecting GlcNAc in lower eukaryotes. 
     In another embodiment, the invention provides a method for producing a human-like glycoprotein in a lower eukaryote (e.g.,  P. pastoris ), wherein the glycoprotein comprises an N-glycan exhibiting a bisecting GlcNAc on a trimannose or trimannosyl (Man 3 GlcNAc 2 ) core structure having at least two GlcNAcs attached to the trimannose core. In this embodiment, Man 3 GlcNAc 2  is reacted with a GnTI activity and then with an N-acetylglucosaminyltransferase II (“GnTII”) activity and a GnTIII activity (in either order) to produce a bisected GlcNAc 3 Man 3 GlcNAc 2  ( FIG. 38 ). It should be appreciated that the bisected trimannosyl core structure of this embodiment may also contain an additional mannosyl group in place of a GlcNAc residue. For example, GlcNAcMan 4 GlcNAc 2  may be reacted with a GnTIII activity to produce a bisected GlcNAc 2 Man 4 GlcNAc 2 . 
     The invention also provides a method for producing a more human-like glycoprotein in a lower eukaryote (e.g.  P. pastoris ), wherein the glycoprotein produced comprises an N-glycan having at least two GlcNAcs attached to a pentamannose core structure (Man 5 GlcNAc 2 ) and which exhibits a bisected N-glycan. Accordingly, in this embodiment, a pentamannose core structure (Man 5 GlcNAc 2 ) is reacted with GnTIII activity to produce a bisected GlcNAcMan 5 GlcNAc 2  and GlcNAc 2 Man 5 GlcNAc 2  structure. 
     In an alternative embodiment, a pentamannose core structure produced via the mutation of och1 and alg3 genes is reacted with α1,2-mannosidase, GnTI, GnTII and GnTIII activities and UDP-GlcNAc to produce a bisected GlcNAc 3 Man 3 GlcNAc 2  glycan ( FIG. 35B ). In another embodiment, a pentamannose core structure is reacted with GnTI and GnTIII activities (in either order or in combination) to produce a bisected GlcNAc 2 Man 5 GlcNAc 2  structure ( FIG. 37 ). 
     In a more preferred embodiment, using the combinatorial DNA library method of the invention, as described below, a pVA53 construct comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP 009571) fused to a catalytically active GnTIII domain from mouse (GnTIII Δ32) is expressed in a  P. pastoris  strain YSH-1 (Example 13) thereby producing N-glycans having a bisected GlcNAc 2 Man 5 GlcNAc 2  structure (Example 20).  FIG. 26  (bottom) displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in the above-mentioned strain, which is designated PBP26 ( FIG. 36 ), exhibiting a predominant peak at 1666 m/z [a], which corresponds to bisected GlcNAc 2 Man 5 GlcNAc 2 . (For comparison,  FIG. 26  (top) displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in strain YSH-1 lacking the pVA53 construct. The predominant peak at 1461 m/z [d] corresponds to the unmodified glycan: GlcNAcMan 5 GlcNAc 2 .) Accordingly, in one embodiment, a host of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 50 mole % of a GlcNAc 2 Man 5 GlcNAc 2  or at least 50 mole % of a GlcNAc 2 Man 3 GlcNAc 2  structure having a bisecting GlcNAc. The mole percent of the glycans is in reference to percent of total neutral glycans as detected by MALDI-TOF. It is understood that if, for example, GlcNAc 2 Man 3 GlcNAc 2  having a bisecting GlcNAc is produced at 20% and GlcNAc 3 Man 3 GlcNAc 2  is produced at 25% on a target protein, the total amount of transiently produced GlcNAc 2 Man 3 GlcNAc 2  having a bisecting GlcNAc is 45%, because GlcNAc 3 Man 3 GlcNAc 2  is a product of a GlcNAc 2 Man 3 GlcNAc 2  having a bisecting GlcNAc further reacted with GnTII. 
     Similarly, in another embodiment, a pVA55 construct comprising the  S. cerevisiae  MNN2(1) leader (GenBank Accession No. NP — 009571) fused to a catalytically active GnTIII domain from mouse (GnTIII Δ32) is expressed in a  P. pastoris  strain (YSH-1) thereby producing N-glycans GlcNAcMan 5 GlcNAc 2  and bisected N-glycans GlcNAc 2 Man 5 GlcNAc 2  structure. As shown in  FIG. 27  (bottom), these structures correspond to peaks at 1463 m/z and 1667 m/z, respectively. (For comparison,  FIG. 27  (top) displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in strain YSH-1 lacking the pVA53 construct. The predominant peak corresponds to unmodified GlcNAcMan 5 GlcNAc 2  at 1461 m/z [d].) Accordingly, in another embodiment, a host of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 20 mole % of a GlcNAc 2 Man 5 GlcNAc 2  or at least 20 mole % of a GlcNAc 2 Man 3 GlcNAc 2  structure having a bisecting GlcNAc. 
     In an even more preferred embodiment, a pVA53 construct comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP 009571) fused to a catalytically active GnTIII domain from mouse (GnTIII 032) is expressed in a  P. pastoris  strain YSH-44 (Example 15) thereby producing N-glycans having a bisected GlcNAc 3 Man 3 GlcNAc 2  structure (Example 20).  FIG. 30  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in the above-mentioned strain designated as YSH-57, exhibiting a predominant peak at 1542 m/z [y], which corresponds to the bisected glycan GlcNAc 3 Man 3 GlcNAc 2 . (For comparison,  FIG. 29  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in strain YSH-44 lacking the pVA53 construct. The predominant peak at 1356 m/z [x] in  FIG. 29  corresponds to the unmodified glycan: GlcNAc 2 Man 3 GlcNAc 2 .) Accordingly, in one embodiment, a host of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 80 mole % of a GlcNAc 3 Man 3 GlcNAc 2  structure having a bisecting GlcNAc. The mole percent of the glycans is in reference to percent of total neutral glycans as detected by MALDI-TOF. 
     Alternatively, in another embodiment, a pVA53 construct comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP 009571) fused to a catalytically active GnTIII domain from mouse (GnTIII Δ32) is expressed in a  P. pastoris  strain (PBP6-5) (Example 11) thereby producing N-glycans having a GlcNAc 2 Man 3 GlcNAc 2  and a bisected GlcNAc 3 Man 3 GlcNAc 2  structure. As shown in  FIG. 32 , these structures correspond to peaks at 1340 m/z and 1543 m/z, respectively. Accordingly, in another embodiment, a host of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 20 mole % of a GlcNAc 3 Man 3 GlcNAc 2  structure having a bisecting GlcNAc in an alg3 mutant host cell. 
     The invention provides methods for producing a human-like glycoprotein in a lower eukaryote, wherein the glycoprotein comprises a Man 5 GlcNAc 2  core structure or a Man 3 GlcNAc 2  core structure, and wherein the core structure is further modified by two or more GlcNAcs. In some embodiments of the invention, 10% or more of the core structures are modified by the two or more GlcNAcs. In other preferred embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80% or even more of the core structures are so modified. In a highly preferred embodiment, one of the GlcNAcs is a bisecting GlcNAc. 
     In another aspect of the invention, a combinatorial nucleic acid library which encodes at least one GnTIII catalytic domain is used to express a GnTIII activity in a lower eukaryotic host cell (Example 18). Preferably, a library of the invention comprises a sublibrary of leader sequences fused in frame to a single nucleic acid molecule or a sublibrary of nucleic acid molecules comprising GnTIII sequences, one or more of which encode a catalytic domain having GnTIII activity in the host cell. Alternatively, a single nucleic acid molecule or a sublibrary of nucleic acid molecules comprising leader sequences is fused in frame to a sublibrary of nucleic acid molecules comprising GnTIII sequences, one or more of which encode a catalytic domain having GnTIII activity in the host cell. (See below.) Expression of these and other such combinatorial libraries is performed in a host cell which expresses a target glycoprotein whose N-glycan structures are analyzed to determine whether and how much GnTIII is expressed. A wide range of catalytically active GnTIII enzymes may be produced in a host cell using the methods and libraries of the invention. It is this aspect of the invention that allows a skilled artisan to create and delinate between GnTIII enzymes having little or no activity and those enzymes that are actively expressed and which produce predominant levels of a desired bisected oligosaccharide intermediate such as GlcNAc 2 Man 5 GlcNAc 2 , GlcNAc 3 Man 3 GlcNAc 2  or GlcNAc 2 Man 3 GlcNAc 2  in the host cells. 
     As described further below, the proper targeting of an enzyme responsible for a given step in the glycosylation pathway to the appropriate subcellular location and the sufficiency of the enzyme&#39;s activity at the particular pH of that subcellular location are important factors in the production of glycoproteins having N-glycans with the desired structures. The use of combinatorial libraries of fusion proteins to generate diverse populations of enzyme chimeras and the screening of these libraries in transformed cells provides a powerful method to identify host strains with the activity of interest in the appropriate location. In preferred embodiments of the invention, the enzyme activity is located such that an N-glycan-containing glycoprotein expressed in the cell is capable of reacting with the activity during the secretion process. 
     Not all combinations of leader/catalytic domains produce desired enzyme activities however. A wide variety of leader/catalytic domain combinations is created, only a few of which may be useful in producing the presently desired intermediates. The present invention, nevertheless, encompasses even those combinations that do not presently exhibit a desired enzymatic activity in the exemplified host cell.  FIG. 28  (bottom) shows a pVB51 construct comprising the  K. lactis  GNT(s) leader (GenBank Accession No. AF106080) fused to a catalytically active GnTIII domain from mouse (GnTIII Δ32) expressed in a  P. pastoris  strain YSH-1, which does not readily exhibit GnTIII activity. (For comparison,  FIG. 28  (top) displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in strain YSH-1 lacking the pVA53 construct. The predominant peak corresponds to unmodified GlcNAcMan 5 GlcNAc 2  at 1461 m/z.) The predominant peak in  FIG. 28  (bottom) at 1463 m/z, which correlates to the mass of GlcNAcMan 5 GlcNAc 2 , is observed. A second peak at 1726 m/z, which does not correlate to the mass of GlcNAc 2 Man 5 GlcNAc 2  is also observed. It is contemplated that these and other such combinations may be useful, with or without slight modifications using techniques well known in the art, when they are expressed, e.g., in other host cells including those which have been modified to produce human-like glycoforms. 
     The use of combinatorial libraries to generate diverse populations of enzyme chimeras and the screening of these libraries in transformed cells further allows strains to be identified in which the enzyme activity is substantially intracellular. Example 6, below, provides an example of assay conditions useful for measuring extracellular α-1,2-mannosidase activity. Examples 22 and 23 also provide examples of assays for glycosyltransferase activity (GnTIII) in the medium. See also Table 9, below, and Choi et al. (2003)  Proc. Natl. Acad. Sci. U.S.A.  100(9):5022-27. For purposes of the invention, an enzyme activity is substantially intracellular when less than 10% of the enzyme activity is measurable in the extracellular medium. 
     As described in Examples 11, 12, 13, 14, 15, and 19-21, a host cell may be engineered by the expression of appropriate glycosyltransferases (e.g., N-acetylglucosaminyltransferase) to produce N-glycans having the desired carbohydrate structures (e.g., GlcNAc 2 Man 3 GlcNAc 2 , GlcNAc 3 Man 3 GlcNAc 2 ). Expression of GnTs in the host cell (e.g., by targeting a nucleic acid molecule or a library of nucleic acid molecules as described below and in Choi et al. (2003)  Proc. Natl. Acad. Sci. U.S.A.  100(9):5022-27 and WO 02/00879) enables the modified host cell to produce N-glycans having the bisecting GlcNAc on the middle mannose. These structures may be processed further using the methods of the invention to produce human-like N-glycans on proteins which enter the secretion pathway of the host cell. 
     In a more preferred embodiment, co-expression of appropriate UDP-sugar-transporter(s) and -transferase(s) will cap the terminal α-1,6 and α-1,3 residues as well as the middle mannose with GlcNAc, resulting in the precursor for mammalian-type complex (e.g. GlcNAc 3 Man 3 GlcNAc 2 ) and hybrid N-glycosylation. These peptide-bound N-linked oligosaccharide chains then serve as a precursor for further modification to a mammalian-type oligosaccharide structure. Subsequent expression of galactosyl-tranferases and genetically engineering the capacity to transfer sialylic acid to the termini (see  FIG. 1B ) will produce a mammalian-type (e.g., human-like) N-glycan structure. 
     Engineering or Selecting Hosts Having N-Acetylglucosaminyltransferase IV, V or VI Activity 
     The present invention provides novel lower eukaryotic hosts having N-acetylglucosaminyltransferase activity that catalyze the formation of a GlcNAcβ1,4 or a GlcNAcβ1,6 glycosidic linkage on the Manα1,6 arm and/or Manα1,3 arm of an oligosaccharide substrate (e.g., GlcNAc 2 Man 3 GlcNAc 2 ) in the presence of a sugar nucleotide UDP-GlcNAc. Transfer of the GlcNAc residues is generally preferred in the presence of a UDP-GlcNAc transporter. The present invention provides recombinant nucleic acid molecules encoding proteins having N-acetylglucosaminyltransferase activity and methods for expressing active enzyme in the yeast secretory pathway. In addition, the present invention provides oligosaccharide structures produced from the transformed hosts that are useful for therapeutic administration. By catalyzing the transfer of the sugar GlcNAc from UDP-GlcNAc onto the oligosaccharide substrates by an N-acetylglucosaminyltransferase activity, multiantennary glycoforms are formed on a protein, which are then extended by galactosyltransferase and sialyltransferases. 
     The invention additionally provides a method for producing a human-like glycoprotein in a lower eukaryotic host cell by expressing N-acetylglucosaminyltransferase IV, V, VI or IX activities (including a full-length enzyme, homologs, variants, derivatives, and catalytically active fragments thereof). In one embodiment, a host cell (e.g.,  P. pastoris ) is engineered to produce more human-like N-glycans, e.g., by activation of an N-acetylglucosaminyltransferase IV, V, VI, IX activities or by expression from a nucleic acid molecule encoding N-acetylglucosaminyltransferase IV, V, VI, IX activities ( FIG. 39 ). Using well-known techniques in the art, gene-specific primers are designed to hybridize to homologous regions of members of glycosyltransferase family, such as GnTIV, V, VI, IX gene sequences, which are readily available in the art (e.g., Genbank, SwissProt databases) and are PCR amplified. 
     Expression of N-Acetylglucosaminyltransferase IV 
     In a first aspect of the invention, a lower eukaryotic host cell is transformed with a nucleotide sequence encoding for the enzyme N-Acetylglucosaminyltransferase IV (“GnTIV”) that catalyzes the addition of a sugar residue β(1,4)N-acetylglucosamine (“GlcNAc”) on the Manα1,3 arm of the GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,2 Manα1,3) Manβ1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn of an oligosaccharide substrate. The addition of a GlcNAcβ1,4 by GnTIV onto the Manα1,3 arm of the acceptor substrate (e.g. GlcNAc 2 Man 3 GlcNAc 2 ) yields a so-called triantennary N-glycan. 
     In one embodiment, the invention provides a method for producing a human-like glycoprotein in a lower eukaryote (e.g.,  P. pastoris ), wherein the glycoprotein comprises a triantennary N-glycan structure on an oligosaccharide structure (e.g., GlcNAc 3 Man 3 GlcNAc 2 ). In this embodiment, the oligosaccharide substrate GlcNAcMan 5 GlcNAc 2  (Choi et al., (2003)  Proc Natl Acad Sci USA  2003 Apr. 29; 100(9):5022-7) is trimmed by an α-1,3/α-1,6-mannosidase activity, such as Mannosidase II (Hamilton et al. (2003)  Science  301:1244 producing the substrate GlcNAc 2 Man 3 GlcNAc 2 , which in turn is reacted with an N-acetylglucosaminyltransferase IV activity to produce a triantennary structure: GlcNAc 3 Man 3 GlcNAc 2  ( FIG. 39 ). Two GnTIV isozymes A and B are set forth in  FIGS. 41 and 42 . In a preferred embodiment, a gene fragment encoding for the human GnTIV ( FIG. 42 ) ligated in-frame to nucleotides 1-108 of the  S. cerevisiae  MNN2(s) targeting peptide sequence is introduced and expressed in  P. pastoris  YSH-44 (Example 15). Thus, in certain embodiments, a host cell of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit preferably at least 50, 60, 70, 80, 90 mole % or more of the desired N-glycan structure GlcNAc 3 Man 3 GlcNAc 2 . In an even more preferred embodiment, the oligosaccharide substrate GlcNAc 3 Man 3 GlcNAc 2  is a substrate for galactosyltransfer reaction. Accordingly, the invention provides a GnTIV activity, which catalyzes the transfer of GlcNAc residues onto oligosaccharide substrates forming a GlcNAcα-1,4 glycosidic linkage on the Manα1,3 arm of an oligosaccharide substrate (e.g., GlcNAc 2 Man 3 GlcNAc 2 ) in lower eukaryotes. 
     In a more preferred embodiment, using the combinatorial DNA library method of the invention, pPB144 construct ( FIG. 40A ) comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP — 009571) fused to a catalytically active GnTIV domain from human (GnTIVB Δ104) is expressed in a  P. pastoris  strain YSH-44 (Example 15) thereby producing triantennary N-glycan GlcNAc 3 Man 3 GlcNAc 2  structures (Example 25).  FIG. 47  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in transformed strain designated as PBP43, exhibiting a predominant peak at 1543 m/z [y], which corresponds to triantennary N-glycan structure GlcNAc 3 Man 3 GlcNAc 2 . (See  FIG. 29  for comparison, which displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in YSH-44 cells producing GlcNAc 2 Man 3 GlcNAc 2 .) Accordingly, in certain embodiments, a host cell of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit preferably at least 50 mole % of a GlcNAc 3 Man 3 GlcNAc 2  triantennary structure. In another embodiment, the host cell of the present invention produces the triantennary structure GlcNAc 3 Man 3 GlcNAc 2  catalyzed by GnTIV in the amount that is at least 60, 70, 80, more preferably 90 mole % or greater. Here and throughout, the mole percent of the glycans is in reference to percent of total neutral glycans as detected by MALDI-TOF MS. 
     Expression of N-Acetylglucosaminyltransferase IV and V 
     In another aspect of the invention, a lower eukaryotic host cell that is engineered or selected to produce triantennary oligosaccharide substrate (e.g., GlcNAc 3 Man 3 GlcNAc 2 ), transformed with a nucleic acid encoding for N-acetylglucosaminyltransferase V (“GnTV”) activity which catalyzes the addition of a sugar residue β(1,6)N-acetylglucosamine (“GlcNAc”) on the Manα1,6 arm of the GlcNAc β1,2-Manα1,6 (GlcNAc β1,4 (GlcNAc β1,2) Manα1,3) Man β1,4-GlcNAc β1,4-GlcNAcβ 1,4-Asn of an oligosaccharide substrate. The addition of a GlcNAcβ1,6 by GnTV onto the acceptor substrate (e.g. GlcNAc 3 Man 3 GlcNAc 2 ) in the presence of GlcNAc residues yields a tetraantennary N-glycan: GlcNAc 4 Man 3 GlcNAc 2 . Preferably, the GnTV activity of the present invention is expressed in a lower eukaryotic host cell producing triantennary glycans, for example in  P. pastoris  PBP43 in which the GnTV activity catalyzes the transfer of a GlcNAc residue onto the Manα1,6 arm of the oligosaccharide substrate GlcNAc 3 Man 3 GlcNAc 2  forming a GlcNAcβ 1,6 glycosidic linkage. 
     In one embodiment, using the combinatorial DNA library method of the invention,  P. pastoris  YSH-44 is transformed with the GnTIVB/ S. cerevisiae  MNN2(s) and GnTV/ S. cerevisiae  MNN2(s) fusion constructs.  FIG. 49  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in transformed strain designated as PBP46, exhibiting a predominant peak at 1747 m/z [z], which corresponds to tetraantennary N-glycan structure GlcNAc 4 Man 3 GlcNAc 2  and a residual peak at 1543 m/z [y], which corresponds to triantennary N-glycan structure GlcNAc 3 Man 3 GlcNAc 2 . 
     In another embodiment, using the combinatorial DNA library method of the invention,  P. pastoris  YSH-44 is transformed with a different combination of enzyme and leader fusion: plasmid pPB128 containing GnTIV A(Δ82)/ S. cerevisiae  MNN2(s) and plasmid pPB140 containing GnTV(Δ145)/ S. cerevisiae  MNN2(s) fusion constructs.  FIG. 50  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in transformed strain designated as PBP94, exhibiting a predominant peak at 1743 m/z [z], which corresponds to tetraantennary N-glycan structure GlcNAc 4 Man 3 GlcNAc 2 . 
     Accordingly, in certain embodiments, the present invention provides a host cell characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 50, 60, 70, 80, 90 mole % or more of the desired N-glycan structure GlcNAc 4 Man 3 GlcNAc 2 . In a more preferred embodiment, by expressing the GnTIV and GnTV activities, the host cell produces the desired multiple antennary N-glycan structure. 
     Not all combinations of leader/catalytic domains produce desired enzyme activities, in the production of multiantennary glycan structures. A wide variety of leader/catalytic domain combinations is created, only a few of which may be useful in producing the presently desired intermediates. The present invention, nevertheless, encompasses even those combinations that do not presently exhibit a desired enzymatic activity in the exemplified host cell. It is contemplated that these and other such combinations may be useful, with or without slight modifications using techniques well known in the art, when they are expressed, e.g., in other host cells including those which have been modified to produce human-like glycoforms. 
     Expression of N-Acetylglucosaminyltransferase V 
     In another embodiment of the invention, a nucleic acid encoding GnTV activity is expressed in host cells producing the core GlcNAc 2 Man 3 GlcNAc 2  structures resulting in the formation of triantennary structures GlcNAc 3 Man 3 GlcNAc 2 . Known GnTV sequence is provided in  FIG. 43 . In one embodiment, using the combinatorial DNA library method of the invention, pPB140 construct ( FIG. 40B ) comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP 009571) fused to a catalytically active GnTV domain from mouse (GnTV Δ145) is expressed in a  P. pastoris  strain YSH-44 (Example 15) thereby producing triantennary N-glycan GlcNAc 3 Man 3 GlcNAc 2  structures (Example 25).  FIG. 48  displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in transformed strain designated as PBP32, exhibiting a predominant peak at 1559 m/z [y], which corresponds to tetraantennary N-glycan structure, and a second peak at 1355 m/z [u] which corresponds to triantennary N-glycan structure GlcNAc 3 Man 3 GlcNAc 2 . (See  FIG. 29  for comparison, which displays the MALDI-TOF spectrum of N-glycans released from a kringle 3 protein expressed in YSH-44 cells producing GlcNAc 2 Man 3 GlcNAc 2 .) Accordingly, in another embodiment, a host of the present invention is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 40 mole % of the triantennary structure: GlcNAc 3 Man 3 GlcNAc 2 . Preferably, the host of the present invention produces at least 50, 60, 70, 80, 90 mole % or greater triantennary structure catalyzed by GnTV. 
     Expression of N-Acetylglucosaminyltransferase VI 
     The invention also provides a lower eukaryotic host cell transformed with a nucleic acid encoding N-acetylglucosaminyltransferase VI (“GnTVI”) activity, which catalyzes the transfer of a sugar residue β(1,4)N-acetylglucosamine on the Manα1,6 arm of the GlcNAc β1,4 (GlcNAc β1,2) Manα1,6 (GlcNAc β1,4 (GlcNAc β1,2) Manα1,3) Man β1,4-GlcNAc β1,4-GlcNAcβ1,4-Asn of an oligosaccharide substrate. The addition of a GlcNAcβ1,4 by GnTVI to an acceptor substrate (e.g. GlcNAc 4 Man 3 GlcNAc 2 ) yields a so-called pentaantennary N-glycan. The addition of the GlcNAc residue forms a GlcNAcβ1,4 glycosidic linkage on the oligosaccharide substrate. In one embodiment, a gene encoding for the GnTVI activity is expressed in a host cell producing pentaantennary N-glycans such as GlcNAc 5 Man 3 GlcNAc 2 . In another embodiment, using the DNA fragment encoding the GnTVI activity set forth in  FIG. 44 , a plasmid construct comprising the  S. cerevisiae  MNN2(s) leader (GenBank Accession No. NP — 009571) fused to a catalytically active GnTVI domain from  Gallus gallus  is expressed in a  P. pastoris  strain YSH-44 thereby producing pentaantennary N-glycan GlcNAc 5 Man 3 GlcNAc 2  structures. The host cell of the present invention is characterized by its ability to produce, at least transiently, N-glycans which produce pentaantennary N-glycans in a detectable moiety. 
     Expression of N-Acetylglucosaminyltransferase IX 
     In another embodiment, the nucleic acid encoding GnTIX activity (e.g., Genbank AN NP 945193) is expressed in a host cell in which the GnTIX activity catalyzes the transfer of GlcNAc residues onto complex glycan acceptor substrates (e.g., GlcNAc 2 Man 3 GlcNAc 2 ) in the absence of GnTIV, GnTV or GnTVI. The nucleic acid encoding GnTIX activity, which normally appears to be expressed exclusively in the brain, has been shown to catalyze the synthesis of a unique N-linked oligosaccharide in CHO mutant cells (Raju et al., (1998)  J Biol Chem  273, 14090-14098). Expression of a recombinant human GnTIX showed GnTV activity, catalyzing the transfer of GlcNAc to the 6-OH position of α1,6 linked mannose arm of the oligosaccharide GlcNAc 2 Man 3 GlcNAc 2 -PA via β1,6 linkage in addition to acting on the α1,3-linked mannose arm ( J Biol Chem.  2003 Oct. 31; 278(44):43102-9). The GnTIX is able to catalyze the transfer of GlcNAc to the 6-OH position of mannose in the sequence GlcNAcβ1,2-Manα1. 
     Accordingly, the present invention provides a method of producing tetraantennary glycan structures in  P. pastoris  using the gene encoding for the GnTIX activity ( FIG. 45 ). Preferably, the introduction and expression of GnTIX activity catalyzes the transfer of GlcNAc residues onto the acceptor substrate GlcNAc 2 Man 3 GlcNAc 2  producing GlcNAc 4 Man 3 GlcNAc 2  and GlcNAc 3 Man 3 GlcNAc 2 . The host cell expression of GnTIX activity catalyzes the transfer of GlcNAc residues to the 6-OH position preferably on both Manα1,3 and Manα1,6 arms of the core GlcNAc 2 Man 3 GlcNAc 2  oligosaccharide substrate producing the tetraantennary structure GlcNAc 4 Man 3 GlcNAc 2 . In one embodiment, a host cell producing the acceptor substrate GlcNAc 2 Man 3 GlcNAc 2 , such as  P. pastoris  YSH-44 is transformed with a plasmid (such as pPB176) comprising the gene encoding for the GnTIX activity (Δ43) fused in frame to the  S. cerevisiae  MNN2(s) leader (Example 29). The host cell is characterized by its ability to produce, at least transiently, N-glycans which exhibit at least 5 mole % or preferably greater GnTIX activity catalyzing the transfer of GlcNAc to the 6-OH position of α1,6 linked mannose arm of the oligosaccharide GlcNAc 2 Man 3 GlcNAc 2  via β1,6 linkage in addition to acting on the α1,3-linked mannose arm of the oligosaccharide substrate. 
     Additionally, the nucleic acid encoding GnTIX activity may be codon optimized for translational efficiency in yeast using well known procedures.  FIG. 46  provides the codon optimized DNA fragment encoding part of the human GnTIX lacking the TM domain (Δ43) synthesized from oligonucleotides using PCR. 
     Variations of Multiple Antennary Glycans 
     The present invention is adapted to produce a variety of multiple antennary glycans which are suitable for therapeutic purposes. It is contemplated that glycans having the same GlcNAcβ-linkages may increase half-life of the proteins. Accordingly, the invention provides a lower eukaryotic host cell comprising N-glycans having at least two GlcNAc residues on either the Manα1,3 or Manα1,6 arm of the trimannose core oligosaccharide intermediate (e.g., Man 3 GlcNAc 2 ). In one embodiment, the lower eukaryotic host cell of comprises at least two GlcNAcβ1,4 residues on the Manα1,3 and Manα1,6 arm of the trimannose core oligosaccharide intermediate (e.g., Man 3 GlcNAc 2 ). In another embodiment, the lower eukaryotic host cell comprises at least two GlcNAcβ1,6 residues on the Manα1,3 and Manα1,6 arm of the trimannose core oligosaccharide intermediate (e.g., Man 3 GlcNAc 2 ). In yet another embodiment, the lower eukaryotic host cell comprises at least two GlcNAcβ1,2 residues on the Manα1,3 and Manα1,6 arm of the trimannose core oligosaccharide intermediate (e.g., Man 3 GlcNAc 2 ). 
     As noted below, while lower eukaryotic host cells are preferred hosts for producing therapeutic proteins using the methods of the invention, the present invention is also useful for modifying N-glycan profiles of glycoproteins made in any eukaryotic host cell, preferably in a non-human (e.g., mammalian) host cell. 
     Host Cells of the Invention 
     A preferred host cell of the invention is a lower eukaryotic cell, e.g., yeast, a unicellular and multicellular or filamentous fungus. However, a wide variety of host cells are envisioned as being useful in the methods of the invention. Plant cells or insect cells, for instance, may be engineered to express a human-like glycoprotein according to the invention. Likewise, a variety of non-human, mammalian host cells may be altered to express more human-like or otherwise altered glycoproteins using the methods of the invention. As one of skill in the art will appreciate, any eukaryotic host cell (including a human cell) may be used in conjunction with a library of the invention to express one or more chimeric proteins which is targeted to a subcellular location, e.g., organelle, in the host cell where the activity of the protein is modified, and preferably is enhanced. Such a protein is preferably—but need not necessarily be—an enzyme involved in protein glycosylation, as exemplified herein. It is envisioned that any protein coding sequence may be targeted and selected for modified activity in a eukaryotic host cell using the methods described herein. 
     Lower eukaryotes that are able to produce glycoproteins having the attached N-glycan Man 5 GlcNAc 2  are particularly useful because (a) lacking a high degree of mannosylation (e.g., greater than 8 mannoses per N-glycan, or especially 30-40 mannoses), they show reduced immunogenicity in humans; and (b) the N-glycan is a substrate for further glycosylation reactions to form an even more human-like glycoform, e.g., by the action of GlcNAc transferase I ( FIG. 1B ; β1,2 GnTI) to form GlcNAcMan 5 GlcNAc 2 . A yield is obtained of greater than 30 mole %, more preferably a yield of 50, 60, 70, 80, 90, or even 100 mole %, glycoproteins with N-glycans having a Man 5 GlcNAc 2  structure. In a preferred embodiment, more than 50% of the Man 5 GlcNAc 2  structure is shown to be a substrate for a GnTI activity and can serve as such a substrate in vivo. 
     Preferred lower eukaryotes of the invention include but are not limited to:  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minutia  (e.g.,  Ogataea minuta, Pichia lindneri ),  Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.  Fusarium gramineum, Fusarium venenatum , and  Neurospora crassa.    
     In each above embodiment, the method is directed to making a host cell in which the oligosaccharide precursors are enriched in Man 5 GlcNAc 2 . These structures are desirable because they may then be processed by treatment in vitro, for example, using the method of Maras and Contreras, U.S. Pat. No. 5,834,251. In a preferred embodiment, however, precursors enriched in Man 5 GlcNAc 2  are processed by at least one further glycosylation reaction in vivo—with glycosidases (e.g., α-mannosidases) and glycosyltransferases (e.g., GnTI)—to produce human-like N-glycans. Oligosaccharide precursors enriched in Man 5 GlcNAc 2 , for example, are preferably processed to those having GlcNAcMan x GlcNAc 2  core structures, wherein X is 3, 4 or 5, and is preferably 3. N-glycans having a GlcNAcMan X GlcNAc 2  core structure where X is greater than 3 may be converted to GlcNAcMan 3 GlcNAc 2 , e.g., by treatment with an α-1,3 and/or α-1,6 mannosidase activity, where applicable. Additional processing of GlcNAcMan 3 GlcNAc 2  by treatment with glycosyltransferases (e.g., GnTII) produces GlcNAc 2 Man 3 GlcNAc 2  core structures which may then be modified, as desired, e.g., by ex vivo treatment or by heterologous expression in the host cell of additional glycosylation enzymes, including glycosyltransferases, sugar transporters and mannosidases (see below), to become human-like N-glycans. 
     Preferred human-like glycoproteins which may be produced according to the invention include those which comprise N-glycans having seven or fewer, or three or fewer, mannose residues; and which comprise one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose. 
     Another preferred non-human host cell of the invention is a lower eukaryotic cell, e.g., a unicellular or filamentous fungus, which is diminished or depleted in the activity of one or more alg gene activities (including an enzymatic activity which is a homolog or equivalent to an alg activity). Another preferred host cell of the invention is diminished or depleted in the activity of one or more enzymes (other than alg activities) that mannosylate the α-1,6 arm of a lipid-linked oligosaccharide structure. 
     While lower eukaryotic host cells are preferred, a wide variety of host cells having the aforementioned properties are envisioned as being useful in the methods of the invention. Plant cells, for instance, may be engineered to express a human-like glycoprotein according to the invention. Likewise, a variety of non-human, mammalian host cells may be altered to express more human-like glycoproteins using the methods of the invention. An appropriate host cell can be engineered, or one of the many such mutants already described in yeasts may be used. A preferred host cell of the invention, as exemplified herein, is a hypermannosylation-minus (OCH1) mutant in  Pichia pastoris  which has further been modified to delete the alg3 gene. 
     The invention additionally provides lower eukaryotic host cells capable of producing glycoproteins having bisected N-glycans, such as bisected GlcNAcMan 5 GlcNAc 2 , GlcNAc 2 Man 5 GlcNAc 2 , GlcNAc 2 Man 3 GlcNAc 2 , and, preferably, GlcNAc 3 Man 3 GlcNAc 2 . In a preferred embodiment of the invention, the host cells comprise a GnTIII activity. In a more preferred embodiment, the host cells further comprise one or more activities selected from: GnTI, GnTII, GnTIV, and GnTV. Preferred host cells express GnTI, GnTII, and GnTIII. Other preferred host cells additionally express GnTIV and/or GnTV. Even more preferably, the one or more GnT activities of the host cells are substantially intracellular. 
     Thus, in preferred embodiments of the invention, host cells comprising the one or more GnT activities produce N-glycans comprising structures, including but not limited to GlcNAcMan 3 GlcNAc 2 , GlcNAcMan 4 GlcNAc 2 , or GlcNAcMan 5 GlcNAc 2 , that are capable of reacting with a GnTIII enzyme activity to produce corresponding bisected N-glycans. The enzyme activities thereby convert glycoproteins containing these N-glycans into forms with new and more desirable properties. Because GnTIII is currently understood to inhibit additional GnT activity in mammalian cells, the skilled artisan should appreciate that sequential glycosylation reaction may or may not be of importance. The present invention contemplates, however, the addition of GnTI and GnTIII in either order or together. It should also be understood that other enzyme activities within the cell, such as, e.g., one or more desired mannosidase activities (e.g., α 1,2 mannosidase, Mannosidase I, Mannosidase II), may act in concert with the GnT activities to generate yet other human-like glycoproteins of interest (see  FIG. 1B ). 
     In a preferred embodiment, a mannosidase II or a catalytically active fragment thereof is introduced into the host cell to trim the α1,3 and α1,6 mannose containing arms of a bisected pentamannose core structure such as GlcNAc 2 Man 5 GlcNAc 2 . The resulting glycans (e.g., bisected GlcNAc 2 Man 4 GlcNAc 2  and GlcNAc 2 Man 3 GlcNAc 2 ) are preferred substrates for subsequent human-like N-glycan modification. 
     In another embodiment of the invention, the host cells comprise a Man 5 GlcNAc 2  core structure or a Man 3 GlcNAc 2  core structure modified by two or more GlcNAcs. It should be understood that either core structure may include further modifications in addition to the modification by GlcNAc. Preferably, 10% or more of the core structures are modified by GlcNAcs. Most preferably, 20%, 30%, 40%, 50%, 60%, 70%, 80% or even more of the core structures contain the GlcNAc modification. 
     Formation of Complex N-Glycans 
     Formation of complex N-glycan synthesis is a sequential process by which specific sugar residues are removed and attached to the core oligosaccharide structure. In higher eukaryotes, this is achieved by having the substrate sequentially exposed to various processing enzymes. These enzymes carry out specific reactions depending on their particular location within the entire processing cascade. This “assembly line” consists of ER, early, medial and late Golgi, and the trans Golgi network all with their specific processing environment. To re-create the processing of human glycoproteins in the Golgi and ER of lower eukaryotes, numerous enzymes (e.g., glycosyltransferases, glycosidases, phosphatases and transporters) have to be expressed and specifically targeted to these organelles, and preferably, in a location so that they function most efficiently in relation to their environment as well as to other enzymes in the pathway. 
     Because one goal of the methods described herein is to achieve a robust protein production strain that is able to perform well in an industrial fermentation process, the integration of multiple genes into the host cell chromosome involves careful planning. As described above, one or more genes which encode enzymes known to be characteristic of non-human glycosylation reactions are preferably deleted. The engineered cell strain is transformed with a range of different genes encoding desired activities, and these genes are transformed in a stable fashion, thereby ensuring that the desired activity is maintained throughout the fermentation process. 
     Any combination of the following enzyme activities may be engineered singly or multiply into the host using methods of the invention: sialyltransferases, mannosidases, fucosyltransferases, galactosyltransferases, GlcNAc transferases, ER and Golgi specific transporters (e.g. syn- and antiport transporters for UDP-galactose and other precursors), other enzymes involved in the processing of oligosaccharides, and enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose and CMP-N-acetylneuraminic acid. Preferably, enzyme activities are introduced on one or more nucleic acid molecules (see also below). Nucleic acid molecules may be introduced singly or multiply, e.g., in the context of a nucleic acid library such as a combinatorial library of the invention. It is to be understood, however, that single or multiple enzymatic activities may be introduced into a host cell in any fashion, including but not limited to protein delivery methods and/or by use of one or more nucleic acid molecules without necessarily using a nucleic acid library or combinatorial library of the invention. 
     Expression of Glycosyltransferases to Produce Complex N-Glycans: 
     With DNA sequence information, the skilled artisan can clone DNA molecules encoding GnT activities (e.g., Example 3, 8, 11, 15, and 18). Using standard techniques well-known to those of skill in the art, nucleic acid molecules encoding GnTI, II, III, IV or V (or encoding catalytically active fragments thereof) may be inserted into appropriate expression vectors under the transcriptional control of promoters and other expression control sequences capable of driving transcription in a selected host cell of the invention, e.g., a fungal host such as  Pichia  sp.,  Kluyveromyces  sp. and  Aspergillus  sp., as described herein, such that one or more of these mammalian GnT enzymes may be actively expressed in a host cell of choice for production of a human-like complex glycoprotein (e.g., Examples 8, 20, and 21). 
     Several individual glycosyltransferases have been cloned and expressed in  S. cerevisiae  (GaIT, GnTI),  Aspergillus nidulans  (GnTI) and other fungi, without however demonstrating the desired outcome of “humanization” on the glycosylation pattern of the organisms (Yoshida et al. (1999)  Glycobiology  9(1):53-8; Kalsner et al. (1995)  Glycoconj. J.  12(3):360-370). It was speculated that the carbohydrate structure required to accept sugars by the action of such glycosyltransferases was not present in sufficient amounts, which most likely contributed to the lack of complex N-glycan formation. 
     A preferred method of the invention provides the functional expression of a GnT, such as GnTI, GnTII, and GnTIII, in the early, medial or late Golgi apparatus, as well as ensuring a sufficient supply of UDP-GlcNAc (e.g., by expression of a UDP-GlcNAc transporter; see Examples below). 
     Methods for Providing Sugar Nucleotide Precursors to the Golgi Apparatus: 
     For a glycosyltransferase to function satisfactorily in the Golgi, the enzyme requires a sufficient concentration of an appropriate nucleotide sugar, which is the high-energy donor of the sugar moiety added to a nascent glycoprotein. In humans, the full range of nucleotide sugar precursors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generally synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. 
     To replicate this process in non-human host cells, such as lower eukaryotes, sugar nucleoside specific transporters have to be expressed in the Golgi to ensure adequate levels of nucleoside sugar precursors (Sommers and Hirschberg (1981)  J. Cell Biol.  91(2):A406-A406; Sommers and Hirschberg (1982)  J. Biol. Chem.  257(18):811-817; Perez and Hirschberg (1987)  Methods in Enzymology  138:709-715). Nucleotide sugars may be provided to the appropriate compartments, e.g., by expressing in the host microorganism an exogenous gene encoding a sugar nucleotide transporter. The choice of transporter enzyme is influenced by the nature of the exogenous glycosyltransferase being used. For example, a GlcNAc transferase may require a UDP-GlcNAc transporter, a fucosyltransferase may require a GDP-fucose transporter, a galactosyltransferase may require a UDP-galactose transporter, and a sialyltransferase may require a CMP-sialic acid transporter. 
     The added transporter protein conveys a nucleotide sugar from the cytosol into the Golgi apparatus, where the nucleotide sugar may be reacted by the glycosyltransferase, e.g., to elongate an N-glycan. The reaction liberates a nucleoside diphosphate or monophosphate, e.g., UDP, GDP, or CMP. Nucleoside monophosphates can be directly exported from the Golgi in exchange for nucleoside triphosphate sugars by an antiport mechanism. Accumulation of a nucleoside diphosphate, however, inhibits the further activity of a glycosyltransferase. As this reaction appears to be important for efficient glycosylation, it is frequently desirable to provide an expressed copy of a gene encoding a nucleotide diphosphatase. The diphosphatase (specific for UDP or GDP as appropriate) hydrolyzes the diphosphonucleoside to yield a nucleoside monophosphate and inorganic phosphate. 
     Suitable transporter enzymes, which are typically of mammalian origin, are described below. Such enzymes may be engineered into a selected host cell using the methods of the invention. 
     In another example, α 2,3- or α 2,6-sialyltransferase caps galactose residues with sialic acid in the trans-Golgi and TON of humans leading to a mature form of the glycoprotein ( FIG. 1B ). To reengineer this processing step into a metabolically engineered yeast or fungus will require (1) α 2,3- or α 2,6-sialyltransferase activity and (2) a sufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi of yeast. To obtain sufficient a 2,3-sialyltransferase activity in the late Golgi, for example, the catalytic domain of a known sialyltransferase (e.g. from humans) has to be directed to the late Golgi in fungi (see above). Likewise, transporters have to be engineered to allow the transport of CMP-N-acetyl neuraminic acid into the late Golgi. There is currently no indication that fungi synthesize or can even transport sufficient amounts of CMP-N-acetyl neuraminic acid into the Golgi. Consequently, to ensure the adequate supply of substrate for the corresponding glycosyltransferases, one has to metabolically engineer the production of CMP-sialic acid into the fungus. 
     UDP-N-Acetylglucosamine 
     The cDNA of human UDP-N-acetylglucosamine transporter, which was recognized through a homology search in the expressed sequence tags database (dbEST), has been cloned (Ishida (1999)  J. Biochem.  126(1):68-77). The mammalian Golgi membrane transporter for UDP-N-acetylglucosamine was cloned by phenotypic correction with cDNA from canine kidney cells (MDCK) of a recently characterized  Kluyveromyces lactis  mutant deficient in Golgi transport of the above nucleotide sugar (Guillen et al. (1998)  Proc. Natl. Acad. Sci. USA  95(14):7888-7892). Results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane (Guillen et al. (1998)  Proc. Natl. Acad. Sci. USA  95(14):7888-7892). 
     Accordingly, one may incorporate the expression of a UDP-GlcNAc transporter in a host cell by means of a nucleic acid construct which may contain, for example: (1) a region by which the transformed construct is maintained in the cell (e.g., origin of replication or a region that mediates chromosomal integration), (2) a marker gene that allows for the selection of cells that have been transformed, including counterselectable and recyclable markers such as ura3 or T-urf13 (Soderholm et al. (2001)  Biotechniques  31(2):306-10) or other well characterized selection-markers (e.g., his4, bla, Sh ble etc.), (3) a gene or fragment thereof encoding a functional UDP-GlcNAc transporter (e.g., from  K. lactis , (Abeijon, (1996)  Proc. Natl. Acad. Sci. U.S.A.  93:5963-5968), or from  H. sapiens  (Ishida et al. (1996)  J. Biochem . (Tokyo) 120(6):1074-8), and (4) a promoter activating the expression of the above mentioned localization/catalytic domain fusion construct library. 
     GDP-Fucose 
     The rat liver Golgi membrane GDP-fucose transporter has been identified and purified by Puglielli and Hirschberg (1999)  J. Biol. Chem.  274(50):35596-35600. The corresponding gene has not been identified, however, N-terminal sequencing can be used for the design of oligonucleotide probes specific for the corresponding gene. These oligonucleotides can be used as probes to clone the gene encoding for GDP-fucose transporter. 
     UDP-Galactose 
     Two heterologous genes, gmal2(+) encoding alpha 1,2-galactosyltransferase (alpha 1,2 GalT) from  Schizosaccharomyces pombe  and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter, have been functionally expressed in  S. cerevisiae  to examine the intracellular conditions required for galactosylation. Correlation between protein galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter, rather than alpha 1,2 GalT played a key role for efficient galactosylation in  S. cerevisiae  (Kainuma (1999)  Glycobiology  9(2):133-141). Likewise, an UDP-galactose transporter from  S. pombe  was cloned (Segawa (1999)  FEBS Letters  451(3):295-298). 
     CMP-N-Acetylneuraminic Acid (CMP-Sialic Acid). 
     Human CMP-sialic acid transporter (hCST) has been cloned and expressed in Lee 8 CHO cells (Aoki et al. (1999)  J. Biochem . (Tokyo) 126(5):940-50; Eckhardt et al. (1997)  Eur. J. Biochem.  248(1):187-92). The functional expression of the murine CMP-sialic acid transporter was achieved in  Saccharomyces cerevisiae  (Berninsone et al. (1997)  J. Biol. Chem.  272(19):12616-9). Sialic acid has been found in some fungi, however it is not clear whether the chosen host system will be able to supply sufficient levels of CMP-Sialic acid. Sialic acid can be either supplied in the medium or alternatively fungal pathways involved in sialic acid synthesis can also be integrated into the host genome. 
     Expression of Diphosphatases: 
     When sugars are transferred onto a glycoprotein, either a nucleoside diphosphate or monophosphate is released from the sugar nucleotide precursors. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction appears to be important for efficient glycosylation, as GDPase from  S. cerevisiae  has been found to be necessary for mannosylation. However, the enzyme only has 10% of the activity towards UDP (Berninsone et al. (1994)  J. Biol. Chem.  269(1):207-211). Lower eukaryotes often do not have UDP-specific diphosphatase activity in the Golgi as they do not utilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.  Schizosaccharomyces pombe , a yeast which adds galactose residues to cell wall polysaccharides (from UDP-galactose), was found to have specific UDPase activity, further suggesting the requirement for such an enzyme (Berninsone et al. (1994)  J. Biol. Chem.  269(1):207-211). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product is important to prevent glycosyltransferase inhibition in the lumen of the Golgi (Khatara et al. (1974)  Eur. J. Biochem.  44:537-560). 
     Methods for Altering N-Glycans in a Host by Expressing a Targeted Enzymatic Activity from a Nucleic Acid Molecule 
     The present invention further provides a method for producing a human-like glycoprotein in a non-human host cell comprising the step of introducing into the cell one or more nucleic acid molecules which encode an enzyme or enzymes for production of the Man 5 GlcNAc 2  carbohydrate structure. In one preferred embodiment, a nucleic acid molecule encoding one or more mannosidase activities involved in the production of Man 5 GlcNAc 2  from Man 8 GlcNAc 2  or Man 9 GlcNAc 2  is introduced into the host. The invention additionally relates to methods for making altered glycoproteins in a host cell comprising the step of introducing into the host cell a nucleic acid molecule which encodes one or more glycosylation enzymes or activities. Preferred enzyme activities are selected from the group consisting of UDP-GlcNAc transferase, UDP-galactosyltransferase, GDP-fucosyltransferase, CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, CMP-sialic acid transporter, and nucleotide diphosphatases. In a particularly preferred embodiment, the host is selected or engineered to express two or more enzymatic activities in which the product of one activity increases substrate levels of another activity, e.g., a glycosyltransferase and a corresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporter activities. In another preferred embodiment, the host is selected or engineered to expresses an activity to remove products which may inhibit subsequent glycosylation reactions, e.g. a UDP- or GDP-specific diphosphatase activity. 
     Preferred methods of the invention involve expressing one or more enzymatic activities from a nucleic acid molecule in a host cell and comprise the step of targeting at least one enzymatic activity to a desired subcellular location (e.g., an organelle) by forming a fusion protein comprising a catalytic domain of the enzyme and a cellular targeting signal peptide, e.g., a heterologous signal peptide which is not normally ligated to or associated with the catalytic domain. The fusion protein is encoded by at least one genetic construct (“fusion construct”) comprising a nucleic acid fragment encoding a cellular targeting signal peptide ligated in the same translational reading frame (“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g., glycosylation enzyme), or catalytically active fragment thereof. 
     The targeting signal peptide component of the fusion construct or protein is preferably derived from a member of the group consisting of: membrane-bound proteins of the ER or Golgi, retrieval signals, Type II membrane proteins, Type I membrane proteins, membrane spanning nucleotide sugar transporters, mannosidases, sialyltransferases, glucosidases, mannosyltransferases and phosphomannosyltransferases. 
     The catalytic domain component of the fusion construct or protein is preferably derived from a glycosidase, mannosidase or a glycosyltransferase activity derived from a member of the group consisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT, Fucosyltransferase and Sialyltransferase. The catalytic domain preferably has a pH optimum within 1.4 pH units of the average pH optimum of other representative enzymes in the organelle in which the enzyme is localized, or has optimal activity at a pH between 5.1 and 8.0. In a preferred embodiment, the catalytic domain encodes a mannosidase selected from the group consisting of  C. elegans  mannosidase IA,  C. elegans  mannosidase IB,  D. melanogaster  mannosidase IA,  H. sapiens  mannosidase IB,  P. citrinum  mannosidase I, mouse mannosidase IA, mouse mannosidase IB,  A. nidulans  mannosidase IA,  A. nidulans  mannosidase IB,  A. nidulans  mannosidase IC, mouse mannosidase II,  C. elegans  mannosidase II,  H. sapiens  mannosidase II, mannosidase Iix, and mannosidase III. 
     Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization 
     In one embodiment of the invention, a human-like glycoprotein is made efficiently in a non-human eukaryotic host cell by introducing into a subcellular compartment of the cell a glycosylation enzyme selected to have a pH optimum similar to the pH optima of other enzymes in the targeted subcellular compartment. For example, most enzymes that are active in the ER and Golgi apparatus of  S. cerevisiae  have pH optima that are between about 6.5 and 7.5 (see Table 3). Because the glycosylation of proteins is a highly evolved and efficient process, the internal pH of the ER and the Golgi is likely also in the range of about 6-8. All previous approaches to reduce mannosylation by the action of recombinant mannosidases in fungal hosts, however, have introduced enzymes that have a pH optimum of around pH 5.0 (Martinet et al. (1998)  Biotech. Letters  20(12): 1171-1177, and Chiba et al. (1998)  J. Biol. Chem.  273(41): 26298-26304). At pH 7.0, the in vitro determined activity of those mannosidases is reduced to less than 10%, which is likely insufficient activity at their point of use, namely, the ER and early Golgi, for the efficient in vivo production of Man 5 GlcNAc 2  on N-glycans. 
     Accordingly, a preferred embodiment of this invention targets a selected glycosylation enzyme (or catalytic domain thereof), e.g., an α-mannosidase, to a subcellular location in the host cell (e.g., an organelle) where the pH optimum of the enzyme or domain is within 1.4 pH units of the average pH optimum of other representative marker enzymes localized in the same organelle(s). The pH optimum of the enzyme to be targeted to a specific organelle should be matched with the pH optimum of other enzymes found in the same organelle to maximize the activity per unit enzyme obtained. Table 3 summarizes the activity of mannosidases from various sources and their respective pH optima. Table 4 summarizes their typical subcellular locations. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Mannosidases and their pH optimum. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 pH 
                   
               
               
                 Source 
                 Enzyme 
                 optimum 
                 Reference 
               
               
                   
               
               
                 
                   Aspergillus saitoi 
                 
                 α-1,2-mannosidase 
                 5.0 
                 Ichishima et al. (1999) 
               
               
                   
                   
                   
                   Biochem. J.  339(Pt 
               
               
                   
                   
                   
                 3): 589-597 
               
               
                 
                   Trichoderma reesei 
                 
                 α-1,2-mannosidase 
                 5.0 
                 Maras et al. (2000) 
               
               
                   
                   
                   
                   J. Biotechnol.  77(2-3): 
               
               
                   
                   
                   
                 255-263 
               
               
                 
                   Penicillium 
                 
                 α-D-1,2- 
                 5.0 
                 Yoshida et al. (1993) 
               
               
                 
                   citrinum 
                 
                 mannosidase 
                   
                   Biochem. J.  290(Pt 
               
               
                   
                   
                   
                 2): 349-354 
               
               
                 
                   C. elegans 
                 
                 α-1,2-mannosidase 
                 5.5 
                 see FIG. 11 
               
               
                 
                   Aspergillus 
                 
                 α-1,2-mannosidase 
                 6.0 
                 Eades and Hintz (2000) 
               
               
                 
                   nidulans 
                 
                   
                   
                   Gene  255(1): 25-34 
               
               
                   Homo sapiens  IA 
                 α-1,2-mannosidase 
                 6.0 
               
               
                 (Golgi) 
               
               
                   Homo sapiens  IB 
                 α-1,2-mannosidase 
                 6.0 
               
               
                 (Golgi) 
               
               
                 
                   Lepidopteran 
                 
                 Type I α-1,2-Man 6 - 
                 6.0 
                 Ren et al. (1995) 
               
               
                 insect cells 
                 mannosidase 
                   
                   Biochem.  34(8): 2489- 
               
               
                   
                   
                   
                 2495 
               
               
                 
                   Homo sapiens 
                 
                 α-D-mannosidase 
                 6.0 
                 Chandrasekaran et al. 
               
               
                   
                   
                   
                 (1984)  Cancer Res.   
               
               
                   
                   
                   
                 44(9): 4059-68 
               
               
                 
                   Xanthomonas 
                 
                 α-1,2,3-mannosidase 
                 6.0 
                 U.S. Pat. No. 6,300,113 
               
               
                 
                   manihotis 
                 
               
               
                 Mouse IB (Golgi) 
                 α-1,2-mannosidase 
                 6.5 
                 Schneikert and 
               
               
                   
                   
                   
                 Herscovics (1994) 
               
               
                   
                   
                   
                   Glycobiology.  4(4): 445- 
               
               
                   
                   
                   
                 50 
               
               
                   Bacillus  sp. 
                 α-D-1,2- 
                 7.0 
                 Maruyama et al. (1994) 
               
               
                 (secreted) 
                 mannosidase 
                   
                 
                   Carbohydrate Res. 
                 
               
               
                   
                   
                   
                 251: 89-98 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment, a particular enzyme or catalytic domain is targeted to a subcellular location in the host cell by means of a chimeric fusion construct encoding a protein comprising a cellular targeting signal peptide not normally associated with the enzymatic domain. Preferably, an enzyme or domain is targeted to the ER, the early, medial or late Golgi, or the trans Golgi apparatus of the host cell. 
     In a more preferred embodiment, the targeted glycosylation enzyme is a mannosidase, glycosyltransferase or a glycosidase. In an especially preferred embodiment, mannosidase activity is targeted to the ER or cis Golgi, where the early reactions of glycosylation occur. While this method is useful for producing a human-like glycoprotein in a non-human host cell, it will be appreciated that the method is also useful more generally for modifying carbohydrate profiles of a glycoprotein in any eukaryotic host cell, including human host cells. 
     Targeting sequences which mediate retention of proteins in certain organelles of the host cell secretory pathway are well-known and described in the scientific literature and public databases, as discussed in more detail below with respect to libraries for selection of targeting sequences and targeted enzymes. Such subcellular targeting sequences may be used alone or in combination to target a selected glycosylation enzyme (or catalytic domain thereof) to a particular subcellular location in a host cell, i.e., especially to one where the enzyme will have enhanced or optimal activity based on pH optima or the presence of other stimulatory factors. 
     When one attempts to trim high mannose structures to yield Man 5 GlcNAc 2  in the ER or the Golgi apparatus of a host cell such as  S. cerevisiae , for example, one may choose any enzyme or combination of enzymes that (1) has a sufficiently close pH optimum (i.e., between pH 5.2 and pH 7.8), and (2) is known to generate, alone or in concert, the specific isomeric Man 5 GlcNAc 2  structure required to accept subsequent addition of GlcNAc by GnTI. Any enzyme or combination of enzymes that is shown to generate a structure that can be converted to GlcNAcMan 5 GlcNAc 2  by GnTI in vitro would constitute an appropriate choice. This knowledge may be obtained from the scientific literature or experimentally. 
     For example, one may determine whether a potential mannosidase can convert Man 8 GlcNAc 2 -2AB (2-aminobenzamide) to Man 5 GlcNAc 2 -AB and then verify that the obtained Man 5 GlcNAc 2 -2AB structure can serve a substrate for GnTI and UDP-GlcNAc to give GlcNAcMan 5 GlcNAc 2  in vitro. Mannosidase IA from a human or murine source, for example, would be an appropriate choice (see, e.g., Example 4). Examples described herein utilize 2-aminobenzamide labeled N-linked oligomannose followed by HPLC analysis to make this determination. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Cellular location and pH optima of various glycosylation- 
               
               
                 related enzymes of  S. cerevisiae.   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Loca- 
                 pH op- 
                   
               
               
                 Gene 
                 Activity 
                 tion 
                 timum 
                 Reference(s) 
               
               
                   
               
               
                 KTR1 
                 α-1,2 
                 Golgi 
                 7.0 
                 Romero et al. 
               
               
                   
                 mannosyltransferase 
                   
                   
                 (1997)  Biochem. J.   
               
               
                   
                   
                   
                   
                 321(Pt 2): 289- 
               
               
                   
                   
                   
                   
                 295 
               
               
                 MNS1 
                 α-1,2-mannosidase 
                 ER 
                 6.5 
                 Lipari et al. 
               
               
                   
                   
                   
                   
                 (1994) 
               
               
                   
                   
                   
                   
                 
                   Glycobiology. 
                 
               
               
                   
                   
                   
                   
                 October; 4(5): 697- 
               
               
                   
                   
                   
                   
                 702 
               
               
                 CWH41 
                 glucosidase I 
                 ER 
                 6.8 
               
               
                 -- 
                 mannosyltransferase 
                 Golgi 
                 7-8 
                 Lehele and 
               
               
                   
                   
                   
                   
                 Tanner (1974) 
               
               
                   
                   
                   
                   
                 
                   Biochim. 
                 
               
               
                   
                   
                   
                   
                 
                   Biophys. Acta 
                 
               
               
                   
                   
                   
                   
                 350(1): 225-235 
               
               
                 KRE2 
                 α-1,2 
                 Golgi 
                 6.5-9.0 
                 Romero et al. 
               
               
                   
                 mannosyltransferase 
                   
                   
                 (1997)  Biochem. J.   
               
               
                   
                   
                   
                   
                 321(Pt 2): 289- 
               
               
                   
                   
                   
                   
                 295 
               
               
                   
               
            
           
         
       
     
     Accordingly, a glycosylation enzyme such as an α-1,2-mannosidase enzyme used according to the invention has an optimal activity at a pH of between 5.1 and 8.0. In a preferred embodiment, the enzyme has an optimal activity at a pH of between 5.5 and 7.5. The  C. elegans  mannosidase enzyme, for example, works well in the methods of the invention and has an apparent pH optimum of about 5.5). Preferred mannosidases include those listed in Table 3 having appropriate pH optima, e.g.  Aspergillus nidulans, Homo sapiens  IA (Golgi),  Homo sapiens  IB (Golgi), Lepidopteran insect cells (IPLB-SF21AE),  Homo sapiens , mouse IB (Golgi),  Xanthomonas manihotis, Drosophila melanogaster  and  C. elegans.    
     An experiment which illustrates the pH optimum for an α-1,2-mannosidase enzyme is described in Example 7. A chimeric fusion protein BB27-2 ( Saccharomyces  MNN10 (s)/ C. elegans  mannosidase IB Δ31), which leaks into the medium was subjected to various pH ranges to determine the optimal activity of the enzyme. The results of the experiment show that the α-1,2-mannosidase has an optimal pH of about 5.5 for its function ( FIG. 11 ). 
     In a preferred embodiment, a single cloned mannosidase gene is expressed in the host organism. However, in some cases it may be desirable to express several different mannosidase genes, or several copies of one particular gene, in order to achieve adequate production of Man 5 GlcNAc 2 . In cases where multiple genes are used, the encoded mannosidases preferably all have pH optima within the preferred range of about 5.1 to about 8.0, or especially between about 5.5 and about 7.5. Preferred mannosidase activities include α-1,2-mannosidases derived from mouse, human, Lepidoptera,  Aspergillus nidulans , or  Bacillus  sp.,  C. elegans, D. melanogaster, P. citrinum, X. laevis  or  A. nidulans.    
     In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNA Library 
     Certain methods of the invention are preferably (but need not necessarily be) carried out using one or more nucleic acid libraries. An exemplary feature of a combinatorial nucleic acid library of the invention is that it comprises sequences encoding cellular targeting signal peptides and sequences encoding proteins to be targeted (e.g., enzymes or catalytic domains thereof, including but not limited to those which mediate glycosylation). 
     In one embodiment, a combinatorial nucleic acid library comprises: (a) at least two nucleic acid sequences encoding different cellular targeting signal peptides; and (b) at least one nucleic acid sequence encoding a polypeptide to be targeted. In another embodiment, a combinatorial nucleic acid library comprises: (a) at least one nucleic acid sequence encoding a cellular targeting signal peptide; and (b) at least two nucleic acid sequences encoding a polypeptide to be targeted into a host cell. As described further below, a nucleic acid sequence derived from (a) and a nucleic acid sequence derived from (b) are ligated to produce one or more fusion constructs encoding a cellular targeting signal peptide functionally linked to a polypeptide domain of interest. One example of a functional linkage is when the cellular targeting signal peptide is ligated to the polypeptide domain of interest in the same translational reading frame (“in-frame”). 
     In a preferred embodiment, a combinatorial DNA library expresses one or more fusion proteins comprising cellular targeting signal peptides ligated in-frame to catalytic enzyme domains. The encoded fusion protein preferably comprises a catalytic domain of an enzyme involved in mammalian- or human-like modification of N-glycans. In a more preferred embodiment, the catalytic domain is derived from an enzyme selected from the group consisting of mannosidases, glycosyltransferases and other glycosidases which is ligated in-frame to one or more targeting signal peptides. The enzyme domain may be exogenous and/or endogenous to the host cell. A particularly preferred signal peptide is one normally associated with a protein that undergoes ER to Golgi transport. 
     The combinatorial DNA library of the present invention may be used for producing and localizing in vivo enzymes involved in mammalian- or human-like N-glycan modification. The fusion constructs of the combinatorial DNA library are engineered so that the encoded enzymes are localized in the ER, Golgi or the trans-Golgi network of the host cell where they are involved in producing particular N-glycans on a glycoprotein of interest. Localization of N-glycan modifying enzymes of the present invention is achieved through an anchoring mechanism or through protein-protein interaction where the localization peptide constructed from the combinatorial DNA library localizes to a desired organelle of the secretory pathway such as the ER, Golgi or the trans Golgi network. 
     An example of a useful N-glycan, which is produced efficiently and in sufficient quantities for further modification by human-like (complex) glycosylation reactions is Man 5 GlcNAc 2 . A sufficient amount of Man 5 GlcNAc 2  is needed on a glycoprotein of interest for further human-like processing in vivo (e.g., more than 30 mole %). The Man 5 GlcNAc 2  intermediate may be used as a substrate for further N-glycan modification to produce GlcNAcMan 5 GlcNAc 2  ( FIG. 1B ; see above). Accordingly, the combinatorial DNA library of the present invention may be used to produce enzymes that subsequently produce GlcNAcMan 5 GlcNAc 2 , or other desired complex N-glycans, in a useful quantity. 
     A further aspect of the fusion constructs produced using the combinatorial DNA library of the present invention is that they enable sufficient and often near complete intracellular N-glycan trimming activity in the engineered host cell. Preferred fusion constructs produced by the combinatorial DNA library of the invention encode a glycosylation enzyme, e.g., a mannosidase, which is effectively localized to an intracellular host cell compartment and thereby exhibits very little and preferably no extracellular activity. The preferred fusion constructs of the present invention that encode a mannosidase enzyme are shown to localize where the N-glycans are modified, namely, the ER and the Golgi. The fusion enzymes of the present invention are targeted to such particular organelles in the secretory pathway where they localize and act upon N-glycans such as Man 8 GlcNAc 2  to produce Man 5 GlcNAc 2  on a glycoprotein of interest. 
     GnTIII fusion constructs generated from a combinatorial DNA library to produce bisected glycans were assayed to determine any extracellular activity. An example of a GnTIII fusion constructs exhibiting in vivo alteration of host cell glycosylation is designated pVA53. After transforming  P. pastoris  YSH-1 with the fusion construct pVA53, the supernatant was tested to detect any ex vivo GnTIII activity.  FIG. 33  shows no apparent change in the standard substrate GlcNAcMan 5 GlcNAc 2  under conditions that would reveal extracellular GnTIII activity in the medium (Example 22). Similarly,  FIG. 34  shows no detectable extracellular GnTIII activity in the medium in  P. pastoris  YSH-57 reacting with the substrate GlcNAc 2 Man 3 GlcNAc 2  (Example 23). 
     Enzymes produced by the combinatorial DNA library of the present invention can modify N-glycans on a glycoprotein of interest as shown for K3 or IFN-β proteins expressed in  P. pastoris , as shown in  FIGS. 5 ,  6 , and  25 - 34  (see also Examples 2, 4, and 18-23). It is, however, appreciated that other types of glycoproteins, without limitation, including erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNase II, α-feto proteins, AAT, rhTBP-1 (onercept, aka TNF Binding protein 1), TACI-Ig (transmembrane activator and calcium modulator and cyclophilin ligand interactor, FSH (follicle stimulating hormone), GM-CSF, GLP-1 w/ and w/o FC (glucagon like protein) IL-1 receptor agonist, sTNFr (Enbrel, aka soluble TNF receptor Fc fusion) ATIII, rhThrombin, glucocerebrosidase and CTLA4-Ig (Cytotoxic T Lymphocyte associated Antigen 4-Ig) may be glycosylated in this way. 
     Constructing a Combinatorial DNA Library of Fusion Constructs: 
     A combinatorial DNA library of fusion constructs features one or more cellular targeting signal peptides (“targeting peptides”) generally derived from N-terminal domains of native proteins (e.g., by making C-terminal deletions). Some targeting peptides, however, are derived from the C-terminus of native proteins (e.g. SEC12). Membrane-bound proteins of the ER or the Golgi are preferably used as a source for targeting peptide sequences. These proteins have sequences encoding a cytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr) which are varied in length. These regions are recognizable by protein sequence alignments and comparisons with known homologs and/or other localized proteins (e.g., comparing hydrophobicity plots). 
     The targeting peptides are indicated herein as short (s), medium (m) and long (l) relative to the parts of a type II membrane. The targeting peptide sequence indicated as short (s) corresponds to the transmembrane domain (tmd) of the membrane-bound protein. The targeting peptide sequence indicated as long (l) corresponds to the length of the transmembrane domain (tmd) and the stem region (sr). The targeting peptide sequence indicated as medium (m) corresponds to the transmembrane domain (tmd) and approximately half the length of the stem region (sr). The catalytic domain regions are indicated herein by the number of nucleotide deletion with respect to its wild-type glycosylation enzyme. 
     Sub-Libraries 
     In some cases a combinatorial nucleic acid library of the invention may be assembled directly from existing or wild-type genes. In a preferred embodiment, the DNA library is assembled from the fusion of two or more sub-libraries. By the in-frame ligation of the sub-libraries, it is possible to create a large number of novel genetic constructs encoding useful targeted protein domains such as those which have glycosylation activities. 
     Catalytic Domain Sub-Libraries Encoding Glycosylation Activities 
     One useful sub-library includes DNA sequences encoding enzymes such as glycosidases (e.g., mannosidases), glycosyltransferases (e.g., fucosyltransferases, galactosyltransferases, glucosyltransferases), GlcNAc transferases and sialyltransferases. Catalytic domains may be selected from the host to be engineered, as well as from other related or unrelated organisms. Mammalian, plant, insect, reptile, algal or fungal enzymes are all useful and should be chosen to represent a broad spectrum of biochemical properties with respect to temperature and pH optima. In a preferred embodiment, genes are truncated to give fragments some of which encode the catalytic domains of the enzymes. By removing endogenous targeting sequences, the enzymes may then be redirected and expressed in other cellular loci. 
     The choice of such catalytic domains may be guided by the knowledge of the particular environment in which the catalytic domain is subsequently to be active. For example, if a particular glycosylation enzyme is to be active in the late Golgi, and all known enzymes of the host organism in the late Golgi have a certain pH optimum, or the late Golgi is known to have a particular pH, then a catalytic domain is chosen which exhibits adequate, and preferably maximum, activity at that pH, as discussed above. 
     Targeting Peptide Sequence Sub-Libraries 
     Another useful sub-library includes nucleic acid sequences encoding targeting signal peptides that result in localization of a protein to a particular location within the ER, Golgi, or trans Golgi network. These targeting peptides may be selected from the host organism to be engineered as well as from other related or unrelated organisms. Generally such sequences fall into three categories: (1) N-terminal sequences encoding a cytosolic tail (ct), a transmembrane domain (tmd) and part or all of a stem region (sr), which together or individually anchor proteins to the inner (lumenal) membrane of the Golgi; (2) retrieval signals which are generally found at the C-terminus such as the HDEL (SEQ ID NO:41) or KDEL (SEQ ID NO:42) tetrapeptide; and (3) membrane spanning regions from various proteins, e.g., nucleotide sugar transporters, which are known to localize in the Golgi. 
     In the first case, where the targeting peptide consists of various elements (ct, tmd and sr), the library is designed such that the ct, the tmd and various parts of the stem region are represented. Accordingly, a preferred embodiment of the sub-library of targeting peptide sequences includes ct, tmd, and/or sr sequences from membrane-bound proteins of the ER or Golgi. In some cases it may be desirable to provide the sub-library with varying lengths of sr sequence. This may be accomplished by PCR using primers that bind to the 5′ end of the DNA encoding the cytosolic region and employing a series of opposing primers that bind to various parts of the stem region. 
     Still other useful sources of targeting peptide sequences include retrieval signal peptides, e.g. the tetrapeptides HDEL or KDEL, which are typically found at the C-terminus of proteins that are transported retrograde into the ER or Golgi. Still other sources of targeting peptide sequences include (a) type II membrane proteins, (b) the enzymes listed in Table 3, (c) membrane spanning nucleotide sugar transporters that are localized in the Golgi, and (d) sequences referenced in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Sources of useful compartmental targeting sequences 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Location 
               
               
                 Gene or 
                   
                   
                 of Gene 
               
               
                 Sequence 
                 Organism 
                 Function 
                 Product 
               
               
                   
               
               
                 MNSI 
                 
                   A. nidulans 
                 
                 α-1,2-mannosidase 
                 ER 
               
               
                 MNSI 
                 
                   A. niger 
                 
                 α-1,2-mannosidase 
                 ER 
               
               
                 MNSI 
                 
                   S. cerevisiae 
                 
                 α-1,2-mannosidase 
                 ER 
               
               
                 GLSI 
                 
                   S. cerevisiae 
                 
                 glucosidase 
                 ER 
               
               
                 GLSI 
                 
                   A. niger 
                 
                 glucosidase 
                 ER 
               
               
                 GLSI 
                 
                   A. nidulans 
                 
                 glucosidase 
                 ER 
               
               
                 HDEL 
                 Universal in 
                 retrieval signal 
                 ER 
               
               
                 at C- 
                 fungi 
               
               
                 terminus 
               
               
                 SEC12 
                 
                   S. cerevisiae 
                 
                 COPII vesicle protein 
                 ER/Golgi 
               
               
                 SEC12 
                 
                   A. niger 
                 
                 COPII vesicle protein 
                 ER/Golgi 
               
               
                 OCH1 
                 
                   S. cerevisiae 
                 
                 1,6-mannosyltransferase 
                 Golgi (cis) 
               
               
                 OCH1 
                 
                   P. pastoris 
                 
                 1,6-mannosyltransferase 
                 Golgi (cis) 
               
               
                 MNN9 
                 
                   S. cerevisiae 
                 
                 1,6-mannosyltransferase 
                 Golgi 
               
               
                   
                   
                 complex 
               
               
                 MNN9 
                 
                   A. niger 
                 
                 undetermined 
                 Golgi 
               
               
                 VAN1 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi 
               
               
                 VAN1 
                 
                   A. niger 
                 
                 undetermined 
                 Golgi 
               
               
                 ANP1 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi 
               
               
                 HOCI 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi 
               
               
                 MNN10 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi 
               
               
                 MNN10 
                 
                   A. niger 
                 
                 undetermined 
                 Golgi 
               
               
                 MNN11 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi (cis) 
               
               
                 MNN11 
                 
                   A. niger 
                 
                 undetermined 
                 Golgi (cis) 
               
               
                 MNT1 
                 
                   S. cerevisiae 
                 
                 1,2-mannosyltransferase 
                 Golgi (cis, 
               
               
                   
                   
                   
                 medial) 
               
               
                 KTR1 
                 
                   P. pastoris 
                 
                 undetermined 
                 Golgi (medial) 
               
               
                 KRE2 
                 
                   P. pastoris 
                 
                 undetermined 
                 Golgi (medial) 
               
               
                 KTR3 
                 
                   P. pastoris 
                 
                 undetermined 
                 Golgi (medial) 
               
               
                 MNN2 
                 
                   S. cerevisiae 
                 
                 1,2-mannosyltransferase 
                 Golgi (medial) 
               
               
                 KTR1 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi (medial) 
               
               
                 KTR2 
                 
                   S. cerevisiae 
                 
                 undetermined 
                 Golgi (medial) 
               
               
                 MNN1 
                 
                   S. cerevisiae 
                 
                 1,3-mannosyltransferase 
                 Golgi (trans) 
               
               
                 MNN6 
                 
                   S. cerevisiae 
                 
                 Phosphomannosyltransferase 
                 Golgi (trans) 
               
               
                 2,6 ST 
                 
                   H. sapiens 
                 
                 2,6-sialyltransferase 
                 trans Golgi 
               
               
                   
                   
                   
                 network 
               
               
                 UDP- 
                 
                   S. pombe 
                 
                 UDP-Gal transporter 
                 Golgi 
               
               
                 Gal T 
               
               
                   
               
            
           
         
       
     
     In any case, it is highly preferred that targeting peptide sequences are selected which are appropriate for the particular enzymatic activity or activities to function optimally within the sequence of desired glycosylation reactions. For example, in developing a modified microorganism capable of terminal sialylation of nascent N-glycans, a process which occurs in the late Golgi in humans, it is desirable to utilize a sub-library of targeting peptide sequences derived from late Golgi proteins. Similarly, the trimming of Man 8 GlcNAc 2  by an α-1,2-mannosidase to give Man 5 GlcNAc 2  is an early step in complex N-glycan formation in humans ( FIG. 1B ). It is therefore desirable to have this reaction occur in the ER or early Golgi of an engineered host microorganism. A sub-library encoding ER and early Golgi retention signals is used. 
     A series of fusion protein constructs (i.e., a combinatorial DNA library) is then constructed by functionally linking one or a series of targeting peptide sequences to one or a series of sequences encoding catalytic domains. In a preferred embodiment, this is accomplished by the in-frame ligation of a sub-library comprising DNA encoding targeting peptide sequences (above) with a sub-library comprising DNA encoding glycosylation enzymes or catalytically active fragments thereof (see below). 
     The resulting library comprises synthetic genes encoding targeting peptide sequence-containing fusion proteins. In some cases it is desirable to provide a targeting peptide sequence at the N-terminus of a fusion protein, or in other cases at the C-terminus. In some cases, targeting peptide sequences may be inserted within the open reading frame of an enzyme, provided the protein structure of individual folded domains is not disrupted. Each type of fusion protein is constructed (in a step-wise directed or semi-random fashion) and optimal constructs may be selected upon transformation of host cells and characterization of glycosylation patterns in transformed cells using methods of the invention. 
     Alteration of Host Cell Glycosylation Using Fusion Constructs from Combinatorial Libraries: 
     The construction of a preferred combinatorial DNA library is illustrated schematically in  FIG. 2  and described in Example 4. The fusion construct may be operably linked to a multitude of vectors, such as expression vectors well-known in the art. A wide variety of such fusion constructs were assembled using representative activities as shown in Table 6. Combinations of targeting peptide/catalytic domains may be assembled for use in targeting mannosidase, glycosyltransferase and glycosidase activities in the ER, Golgi, and the trans Golgi network according to the invention. Surprisingly, the same catalytic domain may have no effect to a very profound effect on N-glycosylation patterns, depending on the type of targeting peptide used (see, e.g., Table 7, Example 4). 
     Mannosidase Fusion Constructs 
     A representative example of a mannosidase fusion construct derived from a combinatorial DNA library of the invention is pFB8, which a truncated  Saccharomyces  SEC12(m) targeting peptide (988-1296 nucleotides of SEC/2 from SwissProt P11655) ligated in-frame to a 187 N-terminal amino acid deletion of a mouse α-mannosidase IA (Genbank AN 6678787). The nomenclature used herein, thus, refers to the targeting peptide/catalytic domain region of a glycosylation enzyme as  Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusion protein localizes in the ER by means of the SEC12 targeting peptide sequence while retaining its mannosidase catalytic domain activity and is capable of producing in vivo N-glycans having a Man 5 GlcNAc 2  structure (Example 4;  FIGS. 6F and 7B ). 
     The fusion construct pGC5,  Saccharomyces  MNS1(m)/mouse mannosidase 113 Δ99, is another example of a fusion construct having intracellular mannosidase trimming activity (Example 4;  FIGS. 5D and 8B ). Fusion construct pBC18-5 ( Saccharomyces  VAN1(s)/ C. elegans  mannosidase IB Δ80) is yet another example of an efficient fusion construct capable of producing N-glycans having a Man 5 GlcNAc 2  structure in vivo. By creating a combinatorial DNA library of these and other such mannosidase fusion constructs according to the invention, a skilled artisan may distinguish and select those constructs having optimal intracellular trimming activity from those having relatively low or no activity. Methods using combinatorial DNA libraries of the invention are advantageous because only a select few mannosidase fusion constructs may produce a particularly desired N-glycan in vivo. 
     In addition, mannosidase trimming activity may be specific to a particular protein of interest. Thus, it is to be further understood that not all targeting peptide/mannosidase catalytic domain fusion constructs may function equally well to produce the proper glycosylation on a glycoprotein of interest. Accordingly, a protein of interest may be introduced into a host cell transfected with a combinatorial DNA library to identify one or more fusion constructs which express a mannosidase activity optimal for the protein of interest. One skilled in the art will be able to produce and select optimal fusion construct(s) using the combinatorial DNA library approach described herein. 
     It is apparent, moreover, that other such fusion constructs exhibiting localized active mannosidase catalytic domains (or more generally, domains of any enzyme) may be made using techniques such as those exemplified in Example 4 and described herein. It will be a matter of routine experimentation for one skilled in the art to make and use the combinatorial DNA library of the present invention to optimize, for example, Man 5 GlcNAc 2  production from a library of fusion constructs in a particular expression vector introduced into a particular host cell. 
     Glycosyltransferase Fusion Constructs 
     Similarly, a glycosyltransferase combinatorial DNA library was made using the methods of the invention. A combinatorial DNA library of sequences derived from glycosyltransferase I (GnTI) activities were assembled with targeting peptides and screened for efficient production in a lower eukaryotic host cell of a GlcNAcMan 5 GlcNAc 2  N-glycan structure on a marker glycoprotein. A fusion construct shown to produce GlcNAcMan 5 GlcNAc 2  (pPB104),  Saccharomyces  MNN9(s)/human GnTI Δ38 was identified (Example 8). A wide variety of such GnTI fusion constructs were assembled (Example 8, Table 10). Other combinations of targeting peptide/GnTI catalytic domains can readily be assembled by making a combinatorial DNA library. It is also apparent to one skilled in the art that other such fusion constructs exhibiting glycosyltransferase activity may be made as demonstrated in Example 8. It will be a matter of routine experimentation for one skilled in the art to use the combinatorial DNA library method described herein to optimize GlcNAcMan 5 GlcNAc 2  production using a selected fusion construct in a particular expression vector and host cell line. 
     As stated above for mannosidase fusion constructs, not all targeting peptide/GnTI catalytic domain fusion constructs will function equally well to produce the proper glycosylation on a glycoprotein of interest as described herein. However, one skilled in the art will be able to produce and select optimal fusion construct(s) using a DNA library approach as described herein. Example 8 illustrates a preferred embodiment of a combinatorial DNA library comprising targeting peptides and GnTI catalytic domain fusion constructs involved in producing glycoproteins with predominantly GlcNAcMan 5 GlcNAc 2  structure. 
     Using Multiple Fusion Constructs to Alter Host Cell Glycosylation 
     In another example of using the methods and libraries of the invention to alter host cell glycosylation, a  P. pastoris  strain with an OCH1 deletion that expresses a reporter protein (K3) was transformed with multiple fusion constructs isolated from combinatorial libraries of the invention to convert high mannose N-glycans to human-like N-glycans (Example 8). First, the mannosidase fusion construct pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187) was transformed into a  P. pastoris  strain lacking 1,6 initiating mannosyltransferases activity (i.e., och1 deletion; Example 1). Second, pPB103 comprising a  K. lactis  MNN2-2 gene (Genbank AN AF106080) encoding an UDP-GlcNAc transporter was constructed to increase further production of GlcNAcMan 5 GlcNAc 2 . The addition of the UDP-GlcNAc transporter increased production of GlcNAcMan 5 GlcNAc 2  significantly in the  P. pastoris  strain as illustrated in  FIG. 10B . Third, pPB104 comprising  Saccharomyces  MNN9 (s)/human GnTI Δ38 was introduced into the strain. This  P. pastoris  strain is referred to as “PBP-3.” (See  FIG. 36 .) 
     It is understood by one skilled in the art that host cells such as the above-described yeast strains can be sequentially transformed and/or co-transformed with one or more expression vectors. It is also understood that the order of transformation is not particularly relevant in producing the glycoprotein of interest. The skilled artisan recognizes the routine modifications of the procedures disclosed herein may provide improved results in the production of the glycoprotein of interest. 
     The importance of using a particular targeting peptide sequence with a particular catalytic domain sequence becomes readily apparent from the experiments described herein. The combinatorial DNA library provides a tool for constructing enzyme fusions that are involved in modifying N-glycans on a glycoprotein of interest, which is especially useful in producing human-like glycoproteins. (Any enzyme fusion, however, may be selected using libraries and methods of the invention.) Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produce K3 with N-glycans of the structure Man 5 GlcNAc 2  as shown in  FIGS. 5D and 5E . This confers a reduced molecular mass to the cleaved glycan compared to the K3 of the parent OCH1 deletion strain, as was detected by MALDI-TOF mass spectrometry in  FIG. 5C . 
     Similarly, the same approach was used to produce another secreted glycoprotein: IFN-β comprising predominantly Man 5 GlcNAc 2 . The Man 5 GlcNAc 2  was removed by PNGase digestion (Papac et al. (1998)  Glycobiology  8:445-454) and subjected to MALDI-TOF as shown in  FIGS. 6A-6F . A single prominent peak at 1254 (m/z) confirms Man 5 GlcNA 2  production on IFN-β in  FIG. 6E  (pGC5) ( Saccharomyces  MNS1(m)/mouse mannosidase IB Δ99) and  6 F (pFB8) ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187). Furthermore, in the  P. pastoris  strain PBP-3 comprising pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187), pPB104 ( Saccharomyces  MNN9 (s)/human GnTI Δ38) and pPB103 ( K. lactis  MNN2-2 gene), the hybrid N-glycan GlcNAcMan 5 GlcNAc 2  [b] was detected by MALDI-TOF ( FIG. 10 ). 
     After identifying transformants with a high degree of mannose trimming, additional experiments were performed to confirm that mannosidase (trimming) activity occurred in vivo and was not predominantly the result of extracellular activity in the growth medium (Example 6;  FIGS. 7-9 ). 
     Although the present invention is exemplified using a  P. pastoris  host organism, it is understood by those skilled in the art that other eukaryotic host cells, including other species of yeast and fungal hosts, may be altered as described herein to produce human-like glycoproteins. The techniques described herein for identification and disruption of undesirable host cell glycosylation genes, e.g. OCH1, is understood to be applicable for these and/or other homologous or functionally related genes in other eukaryotic host cells such as other yeast and fungal strains. As described in Example 9, och1 mnn1 genes were deleted from  K. lactis  to engineer a host cell leading to N-glycans that are completely converted to Man 5 GlcNAc 2  by 1,2-mannosidase ( FIG. 12C ). 
     The MNN1 gene was cloned from  K. lactis  as described in Example 9. The nucleic acid and deduced amino acid sequences of the  K. lactis  MNN1 gene are shown in SEQ ID NOs:43 and 44, respectively. Using gene-specific primers, a construct was made to delete the MNN1 gene from the genome of  K. lactis  (Example 9). Host cells depleted in och1 and mnn1 activities produce N-glycans having a Man 9 GlcNAc 2  carbohydrate structure (see, e.g.,  FIG. 12B ). Such host cells may be engineered further using, e.g., methods and libraries of the invention, to produce mammalian- or human-like glycoproteins. 
     Thus, in another embodiment, the invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of at least forty-five, preferably at least 50, more preferably at least 60 and most preferably 75 or more nucleotide residues of the  K. lactis  MNN1 gene (SEQ ID NO: 43), and homologs, variants and derivatives thereof. The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. Similarly, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided. In addition, also provided are vectors, including expression vectors, which comprise a nucleic acid molecule of the invention, as described further herein. Similarly host cells transformed with the nucleic acid molecules or vectors of the invention are provided. 
     Another aspect of the present invention thus relates to a non-human eukaryotic host strain expressing glycoproteins comprising modified N-glycans that resemble those made by human-cells. Performing the methods of the invention in species other than yeast and fungal cells is thus contemplated and encompassed by this invention. It is contemplated that a combinatorial nucleic acid library of the present invention may be used to select constructs that modify the glycosylation pathway in any eukaryotic host cell system. For example, the combinatorial libraries of the invention may also be used in plants, algae and insects, and in other eukaryotic host cells, including mammalian and human cells, to localize proteins, including glycosylation enzymes or catalytic domains thereof, in a desired location along a host cell secretory pathway. Preferably, glycosylation enzymes or catalytic domains and the like are targeted to a subcellular location along the host cell secretory pathway where they are capable of functioning, and preferably, where they are designed or selected to function most efficiently. 
     Plant and insect cells may also be engineered to alter the glycosylation of expressed proteins using the combinatorial library and methods of the invention. Furthermore, glycosylation in mammalian cells, including human cells, may also be modified using the combinatorial library and methods of the invention. It may be possible, for example, to optimize a particular enzymatic activity or to otherwise modify the relative proportions of various N-glycans made in a mammalian host cell using the combinatorial library and methods of the invention. 
     Examples of modifications to glycosylation which can be affected using a method according to this embodiment of the invention are: (1) engineering a eukaryotic host cell to trim mannose residues from Man 8 GlcNAc 2  to yield a Man 5 GlcNAc 2  N-glycan; (2) engineering eukaryotic host cell to add an N-acetylglucosamine (GlcNAc) residue to Man 5 GlcNAc 2  by action of GlcNAc transferase I; (3) engineering a eukaryotic host cell to functionally express an enzyme such as an N-acetylglucosaminyl Transferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II, fucosyltransferase (FT), galactosyl tranferase (GalT) or a sialyltransferase (ST). 
     By repeating the method, increasingly complex glycosylation pathways can be engineered into a target host, such as a lower eukaryotic microorganism. In one preferred embodiment, the host organism is transformed two or more times with DNA libraries including sequences encoding glycosylation activities. Selection of desired phenotypes may be performed after each round of transformation or alternatively after several transformations have occurred. Complex glycosylation pathways can be rapidly engineered in this manner. 
     Sequential Glycosylation Reactions 
     In a preferred embodiment, such targeting peptide/catalytic domain libraries are designed to incorporate existing information on the sequential nature of glycosylation reactions in higher eukaryotes. Reactions known to occur early in the course of glycoprotein processing require the targeting of enzymes that catalyze such reactions to an early part of the Golgi or the ER. For example, the trimming of Man 8 GlcNAc 2  to Man 5 GlcNAc 2  by mannosidases is an early step in complex N-glycan formation ( FIGS. 1B and 35A ). Because protein processing is initiated in the ER and then proceeds through the early, medial and late Golgi, it is desirable to have this reaction occur in the ER or early Golgi. When designing a library for mannosidase I localization, for example, one thus attempts to match ER and early Golgi targeting signals with the catalytic domain of mannosidase I. 
     Generating Additional Sequence Diversity 
     The method of this embodiment is most effective when a nucleic acid, e.g., a DNA library transformed into the host contains a large diversity of sequences, thereby increasing the probability that at least one transformant will exhibit the desired phenotype. Single amino acid mutations, for example, may drastically alter the activity of glycoprotein processing enzymes (Romero et al. (2000)  J. Biol. Chem.  275(15):11071-4). Accordingly, prior to transformation, a DNA library or a constituent sub-library may be subjected to one or more techniques to generate additional sequence diversity. For example, one or more rounds of gene shuffling, error prone PCR, in vitro mutagenesis or other methods for generating sequence diversity, may be performed to obtain a larger diversity of sequences within the pool of fusion constructs. 
     Expression Control Sequences 
     In addition to the open reading frame sequences described above, it is generally preferable to provide each library construct with expression control sequences, such as promoters, transcription terminators, enhancers, ribosome binding sites, and other functional sequences as may be necessary to ensure effective transcription and translation of the fusion proteins upon transformation of fusion constructs into the host organism. 
     Suitable vector components, e.g., selectable markers, expression control sequences (e.g., promoter, enhancers, terminators and the like) and, optionally, sequences required for autonomous replication in a host cell, are selected as a function of which particular host cell is chosen. Selection criteria for suitable vector components for use in a particular mammalian or a lower eukaryotic host cell are routine. Preferred lower eukaryotic host cells of the invention include  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.  Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa . Where the host is  Pichia pastoris , suitable promoters include, for example, the AOX1, AOX2, GAPDH and P40 promoters. 
     Selectable Markers 
     It is also preferable to provide each construct with at least one selectable marker, such as a gene to impart drug resistance or to complement a host metabolic lesion. The presence of the marker is useful in the subsequent selection of transformants; for example, in yeast the URA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude of selectable markers are known and available for use in yeast, fungi, plant, insect, mammalian and other eukaryotic host cells. 
     Transformation 
     The nucleic acid library is then transformed into the host organism. In yeast, any convenient method of DNA transfer may be used, such as electroporation, the lithium chloride method, or the spheroplast method. In filamentous fungi and plant cells, conventional methods include particle bombardment, electroporation and  agrobacterium  mediated transformation. To produce a stable strain suitable for high-density culture (e.g., fermentation in yeast), it is desirable to integrate the DNA library constructs into the host chromosome. In a preferred embodiment, integration occurs via homologous recombination, using techniques well-known in the art. For example, DNA library elements are provided with flanking sequences homologous to sequences of the host organism. In this manner, integration occurs at a defined site in the host genome, without disruption of desirable or essential genes. 
     In an especially preferred embodiment, library DNA is integrated into the site of an undesired gene in a host chromosome, effecting the disruption or deletion of the gene. For example, integration into the sites of the OCH1, MNN1, or MNN4 genes allows the expression of the desired library DNA while preventing the expression of enzymes involved in yeast hypermannosylation of glycoproteins. In other embodiments, library DNA may be introduced into the host via a nucleic acid molecule, plasmid, vector (e.g., viral or retroviral vector), chromosome, and may be introduced as an autonomous nucleic acid molecule or by homologous or random integration into the host genome. In any case, it is generally desirable to include with each library DNA construct at least one selectable marker gene to allow ready selection of host organisms that have been stably transformed. Recyclable marker genes such as ura3, which can be selected for or against, are especially suitable. 
     Screening and Selection Processes 
     After transformation of the host strain with the DNA library, transformants displaying a desired glycosylation phenotype are selected. Selection may be performed in a single step or by a series of phenotypic enrichment and/or depletion steps using any of a variety of assays or detection methods. Phenotypic characterization may be carried out manually or using automated high-throughput screening equipment. Commonly, a host microorganism displays protein N-glycans on the cell surface, where various glycoproteins are localized. 
     One may screen for those cells that have the highest concentration of terminal GlcNAc on the cell surface, for example, or for those cells which secrete the protein with the highest terminal GlcNAc content. Such a screen may be based on a visual method, like a staining procedure, the ability to bind specific terminal GlcNAc binding antibodies or lectins conjugated to a marker (such lectins are available from E.Y. Laboratories Inc., San Mateo, Calif.), the reduced ability of specific lectins to bind to terminal mannose residues, the ability to incorporate a radioactively labeled sugar in vitro, altered binding to dyes or charged surfaces, or may be accomplished by using a Fluorescence Assisted Cell Sorting (FACS) device in conjunction with a fluorophore labeled lectin or antibody (Guillen et al. (1998)  Proc. Natl. Acad. Sci. USA  95(14):7888-7892). 
     Accordingly, intact cells may be screened for a desired glycosylation phenotype by exposing the cells to a lectin or antibody that binds specifically to the desired N-glycan. A wide variety of oligosaccharide-specific lectins are available commercially (e.g., from EY Laboratories, San Mateo, Calif.). Alternatively, antibodies to specific human or animal N-glycans are available commercially or may be produced using standard techniques. An appropriate lectin or antibody may be conjugated to a reporter molecule, such as a chromophore, fluorophore, radioisotope, or an enzyme having a chromogenic substrate (Guillen et al., 1998 . Proc. Natl. Acad. Sci. USA  95(14): 7888-7892). 
     Screening may then be performed using analytical methods such as spectrophotometry, fluorimetry, fluorescence activated cell sorting, or scintillation counting. In other cases, it may be necessary to analyze isolated glycoproteins or N-glycans from transformed cells. Protein isolation may be carried out by techniques known in the art. In a preferred embodiment, a reporter protein is secreted into the medium and purified by affinity chromatography (e.g. Ni-affinity or glutathione-S-transferase affinity chromatography). In cases where an isolated N-glycan is preferred, an enzyme such as endo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New England Biolabs, Beverly, Mass.) may be used to cleave the N-glycans from glycoproteins. Isolated proteins or N-glycans may then be analyzed by liquid chromatography (e.g., HPLC), mass spectroscopy, or other suitable means. U.S. Pat. No. 5,595,900 teaches several methods by which cells with desired extracellular carbohydrate structures may be identified. In a preferred embodiment, MALDI-TOF mass spectrometry is used to analyze the cleaved N-glycans. 
     Prior to selection of a desired transformant, it may be desirable to deplete the transformed population of cells having undesired phenotypes. For example, when the method is used to engineer a functional mannosidase activity into cells, the desired transformants will have lower levels of mannose in cellular glycoprotein. Exposing the transformed population to a lethal radioisotope of mannose in the medium depletes the population of transformants having the undesired phenotype, i.e., high levels of incorporated mannose (Huffaker and Robbins (1983)  Proc Natl Acad Sci USA.  80(24):7466-70). Alternatively, a cytotoxic lectin or antibody, directed against an undesirable N-glycan, may be used to deplete a transformed population of undesired phenotypes (e.g., Stanley and Siminovitch (1977)  Somatic Cell Genet  3(4):391-405). U.S. Pat. No. 5,595,900 teaches several methods by which cells with a desired extracellular carbohydrate structures may be identified. Repeatedly carrying out this strategy allows for the sequential engineering of more and more complex glycans in lower eukaryotes. 
     To detect host cells having on their surface a high degree of the human-like N-glycan intermediate GlcNAcMan 3 GlcNAc 2 , for example, one may select for transformants that allow for the most efficient transfer of GlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay. This screen may be carried out by growing cells harboring the transformed library under selective pressure on an agar plate and transferring individual colonies into a 96-well microtiter plate. After growing the cells, the cells are centrifuged, the cells resuspended in buffer, and after addition of UDP-GlcNAc and GnTII, the release of UDP is determined either by HPLC or an enzyme linked assay for UDP. Alternatively, one may use radioactively labeled UDP-GlcNAc and GnTII, wash the cells and then look for the release of radioactive GlcNAc by N-acetylglucosaminidase. All this may be carried manually or automated through the use of high throughput screening equipment. Transformants that release more UDP, in the first assay, or more radioactively labeled GlcNAc in the second assay, are expected to have a higher degree of GlcNAcMan 3 GlcNAc 2  on their surface and thus constitute the desired phenotype. Similar assays may be adapted to look at the N-glycans on secreted proteins as well. 
     Alternatively, one may use any other suitable screen such as a lectin binding assay that is able to reveal altered glycosylation patterns on the surface of transformed cells. In this case the reduced binding of lectins specific to terminal mannoses may be a suitable selection tool.  Galantus nivalis  lectin binds specifically to terminal α-1,3 mannose, which is expected to be reduced if sufficient mannosidase II activity is present in the Golgi. One may also enrich for desired transformants by carrying out a chromatographic separation step that allows for the removal of cells containing a high terminal mannose content. This separation step would be carried out with a lectin column that specifically binds cells with a high terminal mannose content (e.g.,  Galantus nivalis  lectin bound to agarose, Sigma, St. Louis, Mo.) over those that have a low terminal mannose content. 
     In addition, one may directly create such fusion protein constructs, as additional information on the localization of active carbohydrate modifying enzymes in different lower eukaryotic hosts becomes available in the scientific literature. For example, it is known that human β1,4-GalTr can be fused to the membrane domain of MNT, a mannosyltransferase from  S. cerevisiae , and localized to the Golgi apparatus while retaining its catalytic activity (Schwientek et al. (1995)  J. Biol. Chem.  270(10):5483-9). If  S. cerevisiae  or a related organism is the host to be engineered one may directly incorporate such findings into the overall strategy to obtain complex N-glycans from such a host. Several such gene fragments in  P. pastoris  have been identified that are related to glycosyltransferases in  S. cerevisiae  and thus could be used for that purpose. 
     Integration Sites 
     As one ultimate goal of this genetic engineering effort is a robust protein production strain that is able to perform well in an industrial fermentation process, the integration of multiple genes into the host (e.g., fungal) chromosome preferably involves careful planning. The engineered strain may likely have to be transformed with a range of different genes, and these genes will have to be transformed in a stable fashion to ensure that the desired activity is maintained throughout the fermentation process. As described herein, any combination of various desired enzyme activities may be engineered into the fungal protein expression host, e.g., sialyltransferases, mannosidases, fucosyltransferases, galactosyltransferases, glucosyltransferases, GlcNAc transferases, ER and Golgi specific transporters (e.g. syn and antiport transporters for UDP-galactose and other precursors), other enzymes involved in the processing of oligosaccharides, and enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose, CMP-N-acetylneuraminic acid. Examples of preferred methods for modifying glycosylation in a lower eukaryotic host cell, such as  Pichia pastoris , are shown in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Some preferred embodiments for modifying glycosylation in a lower eukaroytic microorganism 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Suitable 
               
               
                   
                 Suitable 
                 Suitable Sources of 
                 Suitable 
                 Transporters 
               
               
                 Desired 
                 Catalytic 
                 Localization 
                 Gene 
                 and/or 
               
               
                 Structure 
                 Activities 
                 Sequences 
                 Deletions 
                 Phosphatases 
               
               
                   
               
               
                 Man 5 GlcNAc 2   
                 α-1,2- 
                 Mns1 (N-terminus, 
                 OCH1 
                 none 
               
               
                   
                 mannosidase 
                   S. cerevisiae ) 
                 MNN4 
               
               
                   
                 (murine, 
                 Och1 (N-terminus, 
                 MNN6 
               
               
                   
                 human, 
                   S. cerevisiae , 
               
               
                   
                   Bacillus  sp., 
                   P. pastoris ) 
               
               
                   
                   A. nidulans ) 
                 Ktr1 
               
               
                   
                   
                 Mnn9 
               
               
                   
                   
                 Mnt1 ( S. cerevisiae ) 
               
               
                   
                   
                 KDEL, HDEL 
               
               
                   
                   
                 (C-terminus) 
               
               
                 GlcNAcMan 5 GlcNAc 2   
                 GlcNAc 
                 Och1 (N-terminus, 
                 OCH1 
                 UDP-GlcNAc 
               
               
                   
                 Transferase I, 
                   S. cerevisiae , 
                 MNN4 
                 transporter 
               
               
                   
                 (human, 
                   P. pastoris ) 
                 MNN6 
                 (human, murine, 
               
               
                   
                 murine, rat 
                 KTR1 (N-terminus) 
                   
                   K. lactis ) 
               
               
                   
                 etc.) 
                 Mnn1 (N-terminus, 
                   
                 UDPase 
               
               
                   
                   
                   S. cerevisiae ) 
                   
                 (human) 
               
               
                   
                   
                 Mnt1 (N-terminus, 
               
               
                   
                   
                   S. cerevisiae ) 
               
               
                   
                   
                 GDPase 
               
               
                   
                   
                 (N-terminus, 
               
               
                   
                   
                   S. cerevisiae ) 
               
               
                 GlcNAcMan 3 GlcNAc 2   
                 mannosidase 
                 Ktr1 
                 OCH1 
                 UDP-GlcNAc 
               
               
                   
                 II 
                 Mnn1 (N-terminus, 
                 MNN4 
                 transporter 
               
               
                   
                   
                   S. cerevisiae ) 
                 MNN6 
                 (human, murine, 
               
               
                   
                   
                 Mnt1 (N-terminus, 
                   
                   K. lactis ) 
               
               
                   
                   
                   S. cerevisiae ) 
                   
                 UDPase 
               
               
                   
                   
                 Kre2/Mnt1 
                   
                 (human) 
               
               
                   
                   
                 ( S. cerevisiae ) 
               
               
                   
                   
                 Kre2 ( P. pastoris ) 
               
               
                   
                   
                 Ktr1 ( S. cerevisiae ) 
               
               
                   
                   
                 Ktr1 ( P. pastoris ) 
               
               
                   
                   
                 Mnn1 ( S. cerevisiae ) 
               
               
                 GlcNAc (2-4) - 
                 GlcNAc 
                 Mnn1 (N-terminus, 
                 OCH1 
                 UDP-GlcNAc 
               
               
                 Man 3 GlcNAc 2   
                 Transferase 
                   S. cerevisiae ) 
                 MNN4 
                 transporter 
               
               
                   
                 II, III, IV, V 
                 Mnt1 (N-terminus, 
                 MNN6 
                 (human, murine, 
               
               
                   
                 (human, 
                   S. cerevisiae ) 
                   
                   K. lactis ) 
               
               
                   
                 murine) 
                 Kre2/Mnt1 
                   
                 UDPase 
               
               
                   
                   
                 ( S. cerevisiae ) 
                   
                 (human) 
               
               
                   
                   
                 Kre2 ( P. pastoris ) 
               
               
                   
                   
                 Ktr1 ( S. cerevisiae ) 
               
               
                   
                   
                 Ktr1 ( P. pastoris ) 
               
               
                   
                   
                 Mnn1 ( S. cerevisiae ) 
               
               
                 Gal (1-4) GlcNAc (2-4) - 
                 β-1,4- 
                 Mnn1 (N-terminus, 
                 OCH1 
                 UDP-Galactose 
               
               
                 Man 3 GlcNAc 2   
                 Galactosyltransferase 
                   S. cerevisiae ) 
                 MNN4 
                 transporter 
               
               
                   
                 (human) 
                 Mnt1(N-terminus, 
                 MNN6 
                 (human,  S. pombe ) 
               
               
                   
                   
                   S. cerevisiae ) 
               
               
                   
                   
                 Kre2/Mnt1 
               
               
                   
                   
                 ( S. cerevisiae ) 
               
               
                   
                   
                 Kre2 ( P. pastoris ) 
               
               
                   
                   
                 Ktr1 ( S. cerevisiae ) 
               
               
                   
                   
                 Ktr1 ( P. pastoris ) 
               
               
                   
                   
                 Mnn1 ( S. cerevisiae ) 
               
               
                 NANA (1-4) - 
                 α-2,6- 
                 KTR1 
                 OCH1 
                 CMP-Sialic acid 
               
               
                 Gal (1-4) GlcNAc (2-4) - 
                 Sialyltransferase 
                 MNN1 (N-terminus, 
                 MNN4 
                 transporter 
               
               
                 Man 3 GlcNAc 2   
                 (human) 
                   S. cerevisiae ) 
                 MNN6 
                 (human) 
               
               
                   
                 α-2,3- 
                 MNT1 (N-terminus, 
               
               
                   
                 Sialyltransferase 
                   S. cerevisiae ) 
               
               
                   
                   
                 Kre2/Mnt1 
               
               
                   
                   
                 ( S. cerevisiae ) 
               
               
                   
                   
                 Kre2 ( P. pastoris ) 
               
               
                   
                   
                 Ktr1 ( S. cerevisiae ) 
               
               
                   
                   
                 Ktr1 ( P. pastoris ) 
               
               
                   
                   
                 MNN1 ( S. cerevisiae ) 
               
               
                   
               
            
           
         
       
     
     As any strategy to engineer the formation of complex N-glycans into a host cell such as a lower eukaryote involves both the elimination as well as the addition of particular glycosyltransferase activities, a comprehensive scheme will attempt to coordinate both requirements. Genes that encode enzymes that are undesirable serve as potential integration sites for genes that are desirable. For example, 1,6 mannosyltransferase activity is a hallmark of glycosylation in many known lower eukaryotes. The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been cloned from  S. cerevisiae  and mutations in the gene give rise to a viable phenotype with reduced mannosylation. The gene locus encoding alpha-1,6 mannosyltransferase activity therefore is a prime target for the integration of genes encoding glycosyltransferase activity. In a similar manner, one can choose a range of other chromosomal integration sites that, based on a gene disruption event in that locus, are expected to: (1) improve the cells ability to glycosylate in a more human-like fashion, (2) improve the cells ability to secrete proteins, (3) reduce proteolysis of foreign proteins and (4) improve other characteristics of the process that facilitate purification or the fermentation process itself. 
     Target Glycoproteins 
     The methods described herein are useful for producing glycoproteins, especially glycoproteins used therapeutically in humans. Glycoproteins having specific glycoforms may be especially useful, for example, in the targeting of therapeutic proteins. For example, mannose-6-phosphate has been shown to direct proteins to the lysosome, which may be essential for the proper function of several enzymes related to lysosomal storage disorders such as Gaucher&#39;s, Hunter&#39;s, Hurler&#39;s, Scheie&#39;s, Fabry&#39;s and Tay-Sachs disease, to mention just a few. Likewise, the addition of one or more sialic acid residues to a glycan side chain may increase the lifetime of a therapeutic glycoprotein in vivo after administration. Accordingly, host cells (e.g., lower eukaryotic or mammalian) may be genetically engineered to increase the extent of terminal sialic acid in glycoproteins expressed in the cells. Alternatively, sialic acid may be conjugated to the protein of interest in vitro prior to administration using a sialic acid transferase and an appropriate substrate. Changes in growth medium composition may be employed in addition to the expression of enzyme activities involved in human-like glycosylation to produce glycoproteins more closely resembling human forms (Weikert et al. (1999)  Nature Biotechnology  17, 1116-1121; Werner et al. (1998)  Arzneimittelforschung  48(8):870-880; Andersen and Goochee (1994)  Cur. Opin. Biotechnol.  5:546-549; Yang and Butler (2000)  Biotechnol. Bioengin.  68(4):370-380). Specific glycan modifications to monoclonal antibodies (e.g. the addition of a bisecting GlcNAc) have been shown to improve antibody dependent cell cytotoxicity (Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80), which may be desirable for the production of antibodies or other therapeutic proteins. 
     Therapeutic proteins are typically administered by injection, orally, pulmonary, or other means. Examples of suitable target glycoproteins which may be produced according to the invention include, without limitation: erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsin, DNase II, α-feto proteins, AAT, rhTBP-1 (onercept, aka TNF Binding protein 1), TACI-Ig (transmembrane activator and calcium modulator and cyclophilin ligand interactor, FSH (follicle stimulating hormone), GM-CSF, GLP-1 w/ and w/o FC (glucagon like protein) IL-1 receptor agonist, sTNFr (Enbrel, aka soluble TNF receptor Fc fusion) ATIII, rhThrombin, glucocerebrosidase and CTLA4-Ig (Cytotoxic T Lymphocyte associated Antigen 4-Ig). 
     Expression of GnT-III to Boost Antibody Functionality 
     The addition of N-acetylglucosamine residues to the GlcNAcMan 3 GlcNAc 2  structure by N-acetylglucosaminyltransferases II and III yields a so-called bisected N-glycan GlcNAc 3 Man 3 GlcNAc 2  ( FIG. 15 ). This structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC) (Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80). Re-engineering glycoforms of immunoglobulins expressed by mammalian cells is a tedious and cumbersome task. Especially in the case of GnTIII, where over-expression of this enzyme has been implicated in growth inhibition, methods involving regulated (inducible) gene expression had to be employed to produce immunoglobulins with bisected N-glycans (Umana et al. (1999)  Biotechnol Bioeng.  65(5):542-9; Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80); Umana et al. WO 03/011878; U.S. Pat. No. 6,602,684. 
     Accordingly, in another embodiment, the invention provides systems and methods for producing human-like N-glycans having bisecting N-acetylglucosamine (GlcNAc) on a trimannose or pentamannose core structure. In a preferred embodiment, the invention provides a system and method for producing immunoglobulins having bisected N-glycans. The systems and methods described herein will not suffer from previous problems, e.g., cytotoxicity associated with overexpression of GnTIII or ADCC, as the host cells of the invention are engineered and selected to be viable and preferably robust cells which produce N-glycans having substantially modified human-type glycoforms such as GlcNAc 2 Man 3 GlcNAc 2 . Thus, addition of a bisecting N-acetylglucosamine in a host cell of the invention will have a negligible effect on the growth-phenotype or viability of those host cells. 
     In addition, work by others has shown that there is no linear correlation between GnTIII expression levels and the degree of ADCC. Umana et al. (1999)  Nature Biotechnol.  17:176-80. Thus, finding the optimal expression level in mammalian cells and maintaining it throughout an FDA approved fermentation process seems to be a challenge. However, in cells of the invention, such as fungal cells, finding a promoter of appropriate strength to establish a robust, reliable and optimal GnTIII expression level is a comparatively easy task for one of skill in the art. 
     A host cell such as a yeast strain capable of producing glycoproteins with bisecting N-glycans is engineered according to the invention, by introducing into the host cell a GnTIII activity (Example 12). Preferably, the host cell is transformed with a nucleic acid that encodes GnTIII (see, e.g.,  FIG. 24 ) or a domain thereof having enzymatic activity, optionally fused to a heterologous cell signal targeting peptide (e.g., using the libraries and associated methods of the invention.) Host cells engineered to express GnTIII will produce higher antibody titers than mammalian cells are capable of. They will also produce antibodies with higher potency with respect to ADCC. 
     Antibodies produced by mammalian cell lines transfected with GnTIII have been shown to be as effective as antibodies produced by non-transfected cell-lines, but at a 10-20 fold lower concentration (Davies et al. (2001)  Biotechnol. Bioeng.  74(4):288-94). An increase of productivity of the production vehicle of the invention over mammalian systems by a factor of twenty, and a ten-fold increase of potency will result in a net-productivity improvement of two hundred. The invention thus provides a system and method for producing high titers of an antibody having high potency (e.g., up to several orders of magnitude more potent than what can currently be produced). The system and method is safe and provides high potency antibodies at low cost in short periods of time. Host cells engineered to express GnTIII according to the invention produce immunoglobulins having bisected N-glycans at rates of at least 50 mg/liter/day to at least 500 mg/liter/day. In addition, each immunoglobulin (Ig) molecule (comprising bisecting GlcNAcs) is more potent than the same Ig molecule produced without bisecting GlcNAcs. 
     Production of Multiantennary Structures for Improved Glycoprotein Functionality 
     Synthesis of tetraantennary structures has been found to be important for in vivo biological activity of a variety of proteins such as EPO and α 1 -acid glycoproteins. Takeuchi et al., Proc Natl Acad Sci USA. 1989 October; 86(20):7819-22; Boris et al., Inflammation (1990) 14, 315-323. Pharmacokinetics studies have shown that the bulky structure of the tetraantennary branching prevents EPO from filtering out into the urine. Modification of proteins, for example, with chemical conjugates (e.g., polyethylene glycol), has been devised to retard the clearance of potentially therapeutic glycoproteins. Accordingly, in one embodiment, the present invention provides methods to synthesize glycoproteins comprising multiple antennary structures in lower eukaryotes ( P. pastoris ) that have better in vivo biological activity and are less rapidly cleared than the same glycoprotein having reduced antennarity. In essence, the glycoproteins produced according to the methods of the present invention (see also WO 02/00879 and WO 03/056914 incorporated herein by reference) have improved therapeutic efficacy. 
     The following are examples which illustrate various aspects of the invention. These examples should not be construed as limiting: the examples are included for the purposes of illustration only. 
     Example 1 
     Cloning and Disruption of the OCH1 Gene in  P. pastoris    
     Generation of an OCH1 Mutant of  P. pastoris:    
     A 1215 bp ORF of the  P. pastoris  OCH1 gene encoding a putative α-1,6 mannosyltransferase was amplified from  P. pastoris  genomic DNA (strain X-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides 5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:3) and 5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:4) which were designed based on the  P. pastoris  OCH1 sequence (Japanese Patent Application Publication No. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream of the ORF of the OCH1 gene were amplified from a  P. pastoris  genomic DNA library (Boehm, T. et al. (1999) Yeast 15(7):563-72) using the internal oligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:47) in the OCH1 gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:48) and 5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:49) oligonucleotides in the backbone of the library bearing plasmid lambda ZAP II (Stratagene, La Jolla, Calif.). The resulting 5075 bp fragment was cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9. 
     After assembling a gene knockout construct that substituted the OCH1 reading frame with a HIS4 resistance gene,  P. pastoris  was transformed and colonies were screened for temperature sensitivity at 37° C. OCH1 mutants of  S. cerevisiae  are temperature sensitive and are slow growers at elevated temperatures. One can thus identify functional homologs of OCH1 in  P. pastoris  by complementing an OCH1 mutant of  S. cerevisiae  with a  P. pastoris  DNA or cDNA library. About 20 temperature sensitive strains were further subjected to a colony PCR screen to identify colonies with a deleted ° chi gene. Several och1 deletions were obtained. 
     The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5 kb downstream sequence of OCH1 gene cassette carrying  Pichia  HIS4 gene, was transformed into  P. pastoris  BK1 [GS115 (his4 Invitrogen Corp., San Diego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knock out the wild-type OCH1 gene. The initial screening of transformants was performed using histidine drop-out medium followed by replica plating to select the temperature sensitive colonies. Twenty out of two hundred histidine-positive colonies showed a temperature sensitive phenotype at 37° C. To exclude random integration of pBK9.1 into the  Pichia  genome, the 20 temperature-sensitive isolates were subjected to colony PCR using primers specific to the upstream sequence of the integration site and to HIS4 ORF. Two out of twenty colonies were och1 defective and further analyzed using a Southern blot and a Western blot indicating the functional och1 disruption by the och1 knock-out construct. Genomic DNA were digested using two separate restriction enzymes BglII and ClaI to confirm the och1 knock-out and to confirm integration at the open reading frame. The Western Blot showed och1 mutants lacking a discrete band produced in the GS115 wild type at 46.2 kDa. 
     Example 2 
     Engineering of  P. pastoris  with α-1,2-Mannosidase to Produce Man 5 GlcNAc 2 -Containing IFN-β Precursors 
     An α-1,2-mannosidase is required for the trimming of Man 8 GlcNAc 2  to yield Man 5 GlcNAc 2 , an essential intermediate for complex N-glycan formation. While the production of a Man 5 GlcNAc 2  precursor is essential, it is not necessarily sufficient for the production of hybrid and complex glycans because the specific isomer of Man 5 GlcNAc 2  may or may not be a substrate for GnTI. An och1 mutant of  P. pastoris  is engineered to express secreted human interferon-β under the control of an aox promoter. A DNA library is constructed by the in-frame ligation of the catalytic domain of human mannosidase IB (an α-1,2-mannosidase) with a sub-library including sequences encoding early Golgi and ER localization peptides. The DNA library is then transformed into the host organism, resulting in a genetically mixed population wherein individual transformants each express interferon-β as well as a synthetic mannosidase gene from the library. Individual transformant colonies are cultured and the production of interferon is induced by addition of methanol. Under these conditions, over 90% of the secreted protein is glycosylated interferon-β. 
     Supernatants are purified to remove salts and low-molecular weight contaminants by C 18  silica reversed-phase chromatography. Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produce interferon-β including N-glycans of the structure Man 5 GlcNAc 2 , which has a reduced molecular mass compared to the interferon-β of the parent strain. The purified interferon-β is analyzed by MALDI-TOF mass spectroscopy and colonies expressing the desired form of interferon-β are identified. 
     Example 3 
     Generation of an och1 Mutant Strain Expressing an α-1,2-Mannosidase, GnTI and GnTII for Production of a Human-Like Glycoprotein 
     The 1215 bp open reading frame of the  P. pastoris  OCH1 gene as well as 2685 bp upstream and 1175 bp downstream was amplified by PCR (see also WO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen) and designated pBK9. To create an och1 knockout strain containing multiple auxotrophic markers, 100 μg of pJN329, a plasmid containing an och1::URA3 mutant allele flanked with SfiI restriction sites was digested with SfiI and used to transform  P. pastoris  strain JC308 (Cereghino et al. (2001)  Gene  263:159-169) by electroporation. Following incubation on defined medium lacking uracil for 10 days at room temperature, 1000 colonies were picked and re-streaked. URA + clones that were unable to grow at 37° C., but grew at room temperature, were subjected to colony PCR to test for the correct integration of the och1::URA3 mutant allele. One clone that exhibited the expected PCR pattern was designated YJN153. The Kringle 3 domain of human plasminogen (K3) was used as a model protein. A Neo R  marked plasmid containing the K3 gene was transformed into strain YJN153 and a resulting strain, expressing K3, was named BK64-1. Plasmid pPB 103, containing the  Kluyveromyces lactis  MNN2-2 gene which encodes a Golgi UDP-N-acetylglucosamine transporter was constructed by cloning a blunt BglII-HindIII fragment from vector pDLO2 (Abeijon et al. (1996)  Proc. Natl. Acad. Sci. U.S.A.  93:5963-5968) into BglII and BamHI digested and blunt ended pBLADE-SX containing the  P. pastoris  ADE1 gene (Cereghino et al. (2001)  Gene  263:159-169). This plasmid was linearized with EcoNI and transformed into strain BK64-1 by electroporation and one strain confirmed to contain the MNN2-2 by PCR analysis was named PBP1. 
     A library of mannosidase constructs was generated, comprising in-frame fusions of the leader domains of several type I or type II membrane proteins from  S. cerevisiae  and  P. pastoris  fused with the catalytic domains of several α-1,2-mannosidase genes from human, mouse, fly, worm and yeast sources (see, e.g., WO02/00879, incorporated herein by reference). This library was created in a  P. pastoris  HIS4 integration vector and screened by linearizing with SalI, transforming by electroporation into strain PBP1, and analyzing the glycans released from the K3 reporter protein. One active construct chosen was a chimera of the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fused with a N-terminal deletion of the mouse α-1,2-mannosidase IA gene ( FIG. 3 ), which was missing the 187 nucleotides. A  P. pastoris  strain expressing this construct was named PBP2. 
     A library of GnTI constructs was generated, comprising in-frame fusions of the same leader library with the catalytic domains of GnTI genes from human, worm, frog and fly sources (WO 02/00879). This library was created in a  P. pastoris  ARG4 integration vector and screened by linearizing with AatII, transforming by electroporation into strain PBP2, and analyzing the glycans released from K3. One active construct chosen was a chimera of the first 120 bp of the  S. cerevisiae  MNN9 gene fused to a deletion of the human GnTI gene, which was missing the first 154 bp. A  P. pastoris  strain expressing this construct was named PBP-3. (See also  FIG. 36 .) 
     A library of GnTII constructs was generated, which comprised in-frame fusions of the leader library with the catalytic domains of GnTII genes from human and rat sources (WO 02/00879). This library was created in a  P. pastoris  integration vector containing the NST R  gene conferring resistance to the drug nourseothricin. The library plasmids were linearized with EcoRI, transformed into strain RDP27 by electroporation, and the resulting strains were screened by analysis of the released glycans from purified K3. 
     Materials for the Following Reactions 
     MOPS, sodium cacodylate, manganese chloride, UDP-galactose and CMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA) was from Sigma/Aldrich, Saint Louis, Mo. Recombinant rat α2,6-sialyltransferase from  Spodoptera frugiperda  and β1,4-galactosyltransferase from bovine milk were from Calbiochem (San Diego, Calif.). Protein N-glycosidase F, mannosidases, and oligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearl resin was from TosoHaas. Metal chelating “HisBind” resin was from Novagen (Madison, Wis.). 96-well lysate-clearing plates were from Promega (Madison, Wis.). Protein-binding 96-well plates were from Millipore (Bedford, Mass.). Salts and buffering agents were from Sigma (St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.). 
     Protein Purification 
     Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle 3 was purified from expression media using a C-terminal hexa-histidine tag. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Briefly, a 150 uL (μL) settled volume of resin is poured into the wells of a 96-well lysate-binding plate, washed with 3 volumes of water and charged with 5 volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) and loaded onto the columns. After draining, the columns are washed with 10 volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness by lyophilyzation. 
     Release of N-Linked Glycans 
     The glycans are released and separated from the glycoproteins by a modification of a previously reported method (Papac, et al. A. J. S. (1998)  Glycobiology  8, 445-454). The wells of a 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) are wetted with 100 uL of methanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum after each addition. The dried protein samples are dissolved in 30 uL of RCM buffer and transferred to the wells containing 10 uL of RCM buffer. The wells are drained and washed twice with RCM buffer. The proteins are reduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37° C. The wells are washed three times with 300 uL of water and carboxymethylated by addition of 60 uL of 0.1 M iodoacetic acid for 30 min in the dark at room temperature. The wells are again washed three times with water and the membranes blocked by the addition of 100 uL of 1% PVP 360 in water for 1 hr at room temperature. The wells are drained and washed three times with 300 uL of water and deglycosylated by the addition of 30 uL of 10 mM NH 4 HCO 3  pH 8.3 containing one milliunit of N-glycanase (Glyko). After 16 hours at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness. 
     Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry 
     Molecular weights of the glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer using delayed extraction. The dried glycans from each well were dissolved in 15 uL of water and 0.5 uL spotted on stainless steel sample plates and mixed with 0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowed to dry. 
     Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulse time. The instrument was operated in the delayed extraction mode with a 125 ns delay and an accelerating voltage of 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, the internal pressure was less than 5×10-7 torr, and the low mass gate was 875 Da. Spectra were generated from the sum of 100-200 laser pulses and acquired with a 2 GHz digitizer. Man 5 GlcNAc 2  oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive ion mode. The estimated mass accuracy of the spectra was 0.5%. 
     The mass of the N-glycans eluted from the column is generally associated with a positive ion adduct, which increases the mass by the molecular weight of the positive ion. The most common adducts are H + , Na + and K + . 
     Example 4 
     Engineering of  P. pastoris  to Produce Man 5 GlcNAc 2  as the Predominant N-Glycan Structure Using a Combinatorial DNA Library 
     An och1 mutant of  P. pastoris  (see Examples 1 and 3) was engineered to express and secrete proteins such as the kringle 3 domain of human plasminogen (K3) under the control of the inducible AOXI promoter. The Kringle 3 domain of human plasminogen (K3) was used as a model protein. A DNA fragment encoding the K3 was amplified using Pfu turbo polymerase (Strategene, La Jolla, Calif.) and cloned into EcoRI and XbaI sites of pPICZαA (Invitrogen, Carlsbad, Calif.), resulting in a C-terminal 6-His tag. In order to improve the N-linked glycosylation efficiency of K3 (Hayes et al. 1975  J. Arch. Biochem. Biophys.  171, 651-655), Pro 46  was replaced with Ser 46  using site-directed mutagenesis. The resulting plasmid was designated pBK64. The correct sequence of the PCR construct was confirmed by DNA sequencing. 
     A combinatorial DNA library was constructed by the in-frame ligation of murine α-1,2-mannosidase IB (Genbank AN 6678787) and IA (Genbank AN 6754619) catalytic domains with a sub-library including sequences encoding Cop II vesicle, ER, and early Golgi localization peptides according to Table 6. The combined DNA library was used to generate individual fusion constructs, which were then transformed into the K3 expressing host organism, resulting in a genetically mixed population wherein individual transformants each express K3 as well as a localization signal/mannosidase fusion gene from the library. Individual transformants were cultured and the production of K3 was induced by transfer to a methanol containing medium. Under these conditions, after 24 hours of induction, over 90% of the protein in the medium was K3. The K3 reporter protein was purified from the supernatant to remove salts and low-molecular weight contaminants by Ni-affinity chromatography. Following affinity purification, the protein was desalted by size exclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.) and either directly subjected to MALDI-TOF analysis described below or the N-glycans were removed by PNGase digestion as described below (Release of N-glycans) and subjected to MALDI-TOF analysis Miele et al. (1997)  Biotechnol. Appl. Biochem.  25:151-157. 
     Following this approach, a diverse set of transformants were obtained; some showed no modification of the N-glycans compared to the och1 knockout strain; and others showed a high degree of mannose trimming ( FIGS. 5D and 5E ). Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produced K3 with N-glycans of the structure Man 5 GlcNAc 2 . This confers a reduced molecular mass to the glycoprotein compared to the K3 of the parent och1 deletion strain, a difference which was readily detected by MALDI-TOF mass spectrometry ( FIG. 5 ). Table 7 indicates the relative Man 5 GlcNAc 2  production levels. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 A representative combinatorial DNA library of localization 
               
               
                 sequences/catalytic domains exhibiting relative levels of Man 5 GlcNAc 2  production. 
               
            
           
           
               
               
            
               
                   
                 Targeting peptide sequences 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 MNS1(s) 
                 MNS1(m) 
                 MNS1(l) 
                 SEC12(s) 
                 SEC12(m) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Catalytic 
                 Mouse mannosidase 
                 FB4 
                 FB5 
                 FB6 
                 FB7 
                 FB8 
               
               
                 Domains 
                 1A Δ187 
                 ++ 
                 + 
                 − 
                 ++ 
                 ++++ 
               
               
                   
                 Mouse mannosidase 
                 GB4 
                 GB5 
                 GB6 
                 GB7 
                 GB8 
               
               
                   
                 1B Δ58 
                 ++ 
                 + 
                 + 
                 ++ 
                 + 
               
               
                   
                 Mouse mannosidase 
                 GC4 
                 GC5 
                 GC6 
                 GC7 
                 GC8 
               
               
                   
                 1B Δ99 
                 − 
                 +++ 
                 + 
                 + 
                 + 
               
               
                   
                 Mouse mannosidase 
                 GD4 
                 GD5 
                 GD6 
                 GD7 
                 GD8 
               
               
                   
                 1B Δ170 
                 − 
                 − 
                 − 
                 + 
                 + 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Another combinatorial DNA library of localization 
               
               
                 sequences/catalytic domains exhibiting relative levels of Man 5 GlcNAc 2  production. 
               
            
           
           
               
               
            
               
                   
                 Targeting peptide sequences 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 VAN1(s) 
                 VAN1(m) 
                 VAN1(l) 
                 MNN10(s) 
                 MNN10(m) 
                 MNN10(l) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Catalytic 
                 
                   C. elegans 
                 
                 BC18-5 
                 BC19 
                 BC20 
                 BC27 
                 BC28 
                 BC29 
               
               
                 Domains 
                 mannosidase 
                 +++++ 
                 ++++ 
                 +++ 
                 +++++ 
                 +++++ 
                 +++ 
               
               
                   
                 1B Δ80 
               
               
                   
                 
                   C. elegans 
                 
                 BB18 
                 BB19 
                 BB20 
                 BB18 
                 BB19 
                 BB20 
               
               
                   
                 mannosidase 
                 +++++ 
                 +++++ 
                 ++++ 
                 +++++ 
                 +++++ 
                 ++++ 
               
               
                   
                 1B Δ31 
               
               
                   
               
            
           
         
       
     
     Targeting peptides were selected from MNS I (SwissProt P32906) in  S. cerevisiae  (long, medium and short) (see supra Nucleic Acid Libraries; Combinatorial DNA Library of Fusion Constructs) and SEC12 (SwissProt P11655) in  S. cerevisiae  (988-1140 nucleotides: short) and (988-1296: medium). Although majority of the targeting peptide sequences were N-terminal deletions, some targeting peptide sequences, such as SEC12 were C-terminal deletions. Catalytic domains used in this experiment were selected from mouse mannosidase 1A with a 187 amino acid N-terminal deletion; and mouse mannosidase 1B with a 58, 99 and 170 amino acid deletion. The number of (+)s, as used herein, indicates the relative levels of Man 5 GlcNAc 2  production. The notation (−) indicates no apparent production of Man 5 GlcNAc 2 . The notation (+) indicates less than 10% production of Man 5 GlcNAc 2 , The notation (++) indicates about 10-20% production of Man 5 GlcNAc 2 . The notation with (+++) indicates about 20-40% production of Man 5 GlcNAc 2 . The notation with (++++) indicates about 50% production of Man 5 GlcNAc 2 . The notation with (+++++) indicates greater than 50% production of Man 5 GlcNAc 2 . 
     Table 9 shows relative amount of Man 5 GlcNAc 2  on secreted K3. Six hundred and eight (608) different strains of  P. pastoris , Δoch1 were generated by transforming them with a single construct of a combinatorial genetic library that was generated by fusing nineteen (19) α-1,2 mannosidase catalytic domains to thirty-two (32) fungal ER, and cis-Golgi leaders. 
     
       
         
           
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Amount of Man 5 GlcNAc 2  on secreted K3 
                   
               
               
                 (% of total glycans) 
                 Number of constructs (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 N.D.* 
                 19 
                 (3.1) 
               
               
                  0-10% 
                 341 
                 (56.1) 
               
               
                 10-20% 
                 50 
                 (8.2) 
               
               
                 20-40&amp; 
                 75 
                 (12.3) 
               
               
                 40-60% 
                 72 
                 (11.8) 
               
               
                 More than 60% 
                 51 
                 (8.4)  †   
               
               
                 Total 
                 608 
                 (100) 
               
               
                   
               
               
                 *Several fusion constructs were not tested because the corresponding plasmids could not be propagated in  E. coli  prior to transformation into  P. pastoris . 
               
               
                   †  Clones with the highest degree of Man 5 GlcNAc 2  trimming (30/51) were further analyzed for mannosidase activity in the supernatant of the medium. The majority (28/30) displayed detectable mannosidase activity in the supernatant (e.g. FIG. 4B). Only two constructs displayed high Man 5 GlcNAc 2  levels, while lacking mannosidase activity in the medium (e.g. FIG. 4C). 
               
            
           
         
       
     
     Table 7 shows two constructs pFB8 and pGC5, among others, displaying Man 5 GlcNAc 2 . Table 8 shows a more preferred construct, pBC18-5, a  S. cerevisiae  VAN1(s) targeting peptide sequence (from SwissProt 23642) ligated in-frame to a  C. elegans  mannosidase IB (Genbank AN CAA98114) 80 amino acid N-terminal deletion ( Saccharomyces  Van1(s)/ C. elegans  mannosidase IB Δ80). This fusion construct also produces a predominant Man 5 GlcNAc 2  structure, as shown in  FIG. 5E . This construct was shown to produce greater than 50% Man 5 GlcNAc 2  (+++++). 
     Generation of a Combinatorial Localization/Mannosidase Library: 
     Generating a combinatorial DNA library of α-1,2-mannosidase catalytic domains fused to targeting peptides required the amplification of mannosidase domains with varying lengths of N-terminal deletions from a number of organisms. To approach this goal, the full length open reading frames (ORFs) of α-1,2-mannosidases were PCR amplified from either cDNA or genomic DNA obtained from the following sources:  Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Aspergillus nidulans  and  Penicillium citrinum . In each case, DNA was incubated in the presence of oligonucleotide primers specific for the desired mannosidase sequence in addition to reagents required to perform the PCR reaction. For example, to amplify the ORF of the  M. musculus  α-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC (SEQ ID NO:52) and the 3′-primer TCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:53) were incubated in the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and amplified under the conditions recommended by Stratagene using the cycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification the DNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1 U Taq DNA polymerase (Promega, Madison, Wis.) prior to ligation into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP10 chemically competent  E. coli , as recommended by Invitrogen. The cloned PCR product was confirmed by ABI sequencing using primers specific for the mannosidase ORF. 
     To generate the desired N-terminal truncations of each mannosidase, the complete ORF of each mannosidase was used as the template in a subsequent round of PCR reactions wherein the annealing position of the 5′-primer was specific to the 5′-terminus of the desired truncation and the 3′-primer remained specific for the original 3′-terminus of the ORF. To facilitate subcloning of the truncated mannosidase fragment into the yeast expression vector, pJN347 ( FIG. 2C ) AscI and PacI restriction sites were engineered onto each truncation product, at the 5′- and 3′-termini respectively. The number and position of the N-terminal truncations generated for each mannosidase ORF depended on the position of the transmembrane (TM) region in relation to the catalytic domain (CD). For instance, if the stem region located between the TM and CD was less than 150 bp, then only one truncation for that protein was generated. If, however, the stem region was longer than 150 bp then either one or two more truncations were generated depending on the length of the stem region. 
     An example of how truncations for the  M. musculus  mannosidase IA (Genbank AN 6678787) were generated is described herein, with a similar approach being used for the other mannosidases.  FIG. 3  illustrates the ORF of the  M. musculus  α-1,2-mannosidase IA with the predicted transmembrane and catalytic domains being highlighted in bold. Based on this structure, three 5′-primers were designed (annealing positions underlined in  FIG. 3 ) to generate the Δ65-, Δ105- and Δ187-N-terminal deletions. Using the Δ65 N-terminal deletion as an example the 5′-primer used was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ ID NO:54) (with the AscI restriction site highlighted in bold) in conjunction with the 3′-primer 5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO:55) (with the PacI restriction site highlighted in bold). Both of these primers were used to amplify a 1561 bp fragment under the conditions outlined above for amplifying the full length  M. musculus  mannosidase 1A ORF. Furthermore, like the product obtained for the full length ORF, the truncated product was also incubated with Taq DNA polymerase, ligated into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10 and ABI sequenced. After having amplified and confirmed the sequence of the truncated mannosidase fragment, the resulting plasmid, pCR2.1-Δ65mMannIA, was digested with AscI and PacI in New England Biolabs buffer #4 (Beverly, Mass.) for 16 h at 37° C. In parallel, the pJN347 ( FIG. 2C ) was digested with the same enzymes and incubated as described above. Post-digestion, both the pJN347 ( FIG. 2C ) back-bone and the truncated catalytic domain were gel extracted and ligated using the Quick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommended by the manufacturers, and transformed into chemically competent DH5α cells (Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm the generation of the pJN347-mouse Mannosidase IAΔ65 construct. 
     Having generated a library of truncated α-1,2-mannosidase catalytic domains in the yeast expression vector pJN347 ( FIG. 2C ) the remaining step in generating the targeting peptide/catalytic domain library was to clone in-frame the targeting peptide sequences ( FIG. 2 ). Both the pJN347-mannosidase constructs ( FIG. 2D ) and the pCR2.1TOPO-targeting peptide constructs ( FIG. 2B ) such as were incubated overnight at 37° C. in New England Biolabs buffer #4 in the presence of the restriction enzymes NotI and AscI. Following digestion, both the pJN347-mannosidase back-bone and the targeting peptide regions were gel-extracted and ligated using the Quick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommended by the manufacturers, and transformed into chemically competent DH5α cells (Invitrogen, Carlsbad, Calif.). Subsequently, the pJN347-targeting peptide/mannosidase constructs were ABI sequenced to confirm that the generated fusions were in-frame. The estimated size of the final targeting peptide/alpha-1,2-mannosidase library contains over 1300 constructs generated by the approach described above.  FIG. 2  illustrates construction of the combinatorial DNA library. 
     Engineering a  P. pastoris  OCH1 Knock-Out Strain with Multiple Auxotrophic Markers. 
     The first step in plasmid construction involved creating a set of universal plasmids containing DNA regions of the KEX1 gene of  P. pastoris  (Boehm et al.  Yeast  1999 May; 15(7):563-72) as space holders for the 5′ and 3′ regions of the genes to be knocked out. The plasmids also contained the  S. cerevisiae  Ura-blaster (Alani et al. (1987)  Genetics  116:541-545) as a space holder for the auxotrophic markers, and an expression cassette with a multiple cloning site for insertion of a foreign gene. A 0.9-kb fragment of the  P. pastoris  KEX1-5′ region was amplified by PCR using primers 
                    (SEQ ID NO: 56)       GGC GAGCTCGGCCTACCCGGCC AAGGCTGAGATCATTTGTCCAGCTTCA       GA       and               (SEQ ID NO: 57)       GCCCAC GTCGACGGATCCGTTTAAAC ATCGATTGGAGAGGCTGACACC       GCTACTA            
and  P. pastoris  genomic DNA as a template and cloned into the SacI, SalI sites of pUC19 (New England Biolabs, Beverly, Mass.). The resulting plasmid was cut with BamHI and SalI, and a 0.8-kb fragment of the KEX1-3′ region that had been amplified using primers
 
                    (SEQ ID NO: 58)       CG GGATCCACTAGTATTTAAAT CATATGTGCGAGTGTACAACTCTTCCC       ACATGG       and               (SEQ ID NO: 59)       GGACGC GTCGACGGCCTACCCGGCC GTACGAGGAATTTCTCGG       ATGACTCTTTTC            
was cloned into the open sites creating pJN262. This plasmid was cut with BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani et al. (1987)  Genetics  116:541-545) was inserted in both possible orientations resulting in plasmids pJN263 ( FIG. 4A ) and pJN284 ( FIG. 4B ).
 
     An expression cassette was created with NotI and PacI as cloning sites. The GAPDH promoter of  P. pastoris  was amplified using primers 
                            (SEQ ID NO: 60)           CG GGATCCCTCGAGAGATCT TTTTTGTAGAAATGTCTTGGTGCCT           and                       (SEQ ID NO: 61)           GGACAT GCATGCACTAGTGCGGCCGCCACGTG ATAGTTGTTCA           ATTGATTGAAATAGGGACAA            
and plasmid pGAPZ-A (Invitrogen) as template and cloned into the BamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) ( FIG. 4B ). The resulting plasmid was cut with SpeI and SphI and the CYC1 transcriptional terminator region (“TT”) that had been amplified using primers
 
                            (SEQ ID NO: 62)           CCTT GCTAGCTTAATTAACCGCGG CACGTCCGACGGCGGCCCA           CGGGTCCCA           and           (SEQ ID NO: 63)           GGACAT GCATGCGGATCCCTTAAG A GCCGGC AGCTTGCAAATT           AAAGCCTTCGAGCGTCCC            
and plasmid pPICZ-A (Invitrogen) as a template was cloned into the open sites creating pJN261 ( FIG. 4B ).
 
     A knockout plasmid for the  P. pastoris  OCH1 gene was created by digesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of the OCH1-5′ region, which had been amplified using the primers 
                            (SEQ ID NO: 64)           GAACCAC GTCGACGGCCATTGCGGCC AAAACCTTTTTTCCTATT           CAAACACAAGGCATTGC           and                       (SEQ ID NO: 65)           CTCCAAT ACTAGT CGAAGATTATCTTCTACGGTGCCTGGACTC            
and  P. pastoris  genomic DNA as a template, was cloned into the open sites ( FIG. 4C ). The resulting plasmid was cut with EcoRI and PmeI and a 1.0-kb DNA fragment of the OCH1-3′ region that had been generated using the primers
 
                            (SEQ ID NO: 66)           TGGAAG GTTTAAAC AAAGCTAGAGTAAAATAGATATAGCGAG           ATTAGAGAATG           and                       (SEQ ID NO: 67)           AA GAATTC GGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT           TTCATC            
was inserted to generate pJN298 ( FIG. 4C ). To allow for the possibility to simultaneously use the plasmid to introduce a new gene, the BamHI expression cassette of pJN261 ( FIG. 4B ) was cloned into the unique BamHI site of pJN298 ( FIG. 4C ) to create pJN299 ( FIG. 4E ).
 
     The  P. pastoris  Ura3-blaster cassette was constructed using a similar strategy as described in Lu et al. (1998)  Appl. Microbiol. Biotechnol.  49:141-146. A 2.0-kb PstI, SpeI fragment of  P. pastoris  URA3 was inserted into the PstI, XbaI sites of pUC19 (New England Biolabs, Beverly, Mass.) to create pJN306 ( FIG. 4D ). Then a 0.7-kb SacI, PvuII DNA fragment of the lacZ open reading frame was cloned into the SacI, SmaI sites to yield pJN308 ( FIG. 4D ). Following digestion of pJN308 ( FIG. 4D ) with PstI, and treatment with T4 DNA polymerase, the SacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNA polymerase was inserted generating pJN315 ( FIG. 4D ). The lacZ/URA3 cassette was released by digestion with SacI and SphI, blunt ended with T4 DNA polymerase and cloned into the backbone of pJN299 that had been digested with PmeI and AflII and blunt ended with T4 DNA polymerase. The resulting plasmid was named pJN329 ( FIG. 4E ). 
     A HIS4 marked expression plasmid was created by cutting pJN261 ( FIG. 4F ) with EcoICRI ( FIG. 4F ). A 2.7 kb fragment of the  Pichia pastoris  HIS4 gene that had been amplified using the primers 
                    (SEQ ID NO: 68)       GCCCAA GCCGGCCTTAAG GGATCTCCTGATGACTGACTCACTGATAATA       AAAATACGG       and               (SEQ ID NO: 69)       GGGCGCGT ATTTAAATACTAGT GGATCTATCGAATCTAAATGTAAGTTA       AAATCTCTAA            
cut with NgoMIV and SwaI and then blunt-ended using T4 DNA polymerase, was then ligated into the open site. This plasmid was named pJN337 ( FIG. 4F ). To construct a plasmid with a multiple cloning site suitable for fusion library construction, pJN337 was cut with NotI and PacI and the two oligonucleotides
 
                            (SEQ ID NO: 70)             GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT             and                       (SEQ ID NO: 71)             TAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC              
that had been annealed in vitro were ligated into the open sites, creating pJN347 ( FIG. 4F ).
 
     To create an och1 knockout strain containing multiple auxotrophic markers, 100 μg of pJN329 was digested with SfiI and used to transform  P. pastoris  strain JC308 (Cereghino et al. (2001)  Gene  263:159-169) by electroporation. Following transformation, the URA dropout plates were incubated at room temperature for 10 days. One thousand (1000) colonies were picked and restreaked. All 1000 clones were then streaked onto 2 sets of URA dropout plates. One set was incubated at room temperature, whereas the second set was incubated at 37° C. The clones that were unable to grow at 37° C., but grew at room temperature, were subjected to colony PCR to test for the correct OCH1 knockout. One clone that showed the expected PCR signal (about 4.5 kb) was designated YJN153. 
     Example 5 
     Characterization of the Combinatorial DNA Library 
     Positive transformants screened by colony PCR confirming integration of the mannosidase construct into the  P. pastoris  genome were subsequently grown at room temperature in 50 ml BMGY buffered methanol-complex medium consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10 −5 % biotin, and 1% glycerol as a growth medium) until ° D 600nm  2-6 at which point they were washed with 10 ml BMMY (buffered methanol-complex medium consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10 −5 % biotin, and 1.5% methanol as a growth medium) media prior to induction of the reporter protein for 24 hours at room temperature in 5 ml BMMY. Consequently, the reporter protein was isolated and analyzed as described in Example 3 to characterize its glycan structure. Using the targeting peptides in Table 6, mannosidase catalytic domains localized to either the ER or the Golgi showed significant level of trimming of a glycan predominantly containing Man 8 GlcNAc 2  to a glycan predominantly containing Man 5 GlcNAc 2 . This is evident when the glycan structure of the reporter glycoprotein is compared between that of  P. pastoris  och1 knock-out in  FIGS. 5C and 6C  and the same strain transformed with  M. musculus  mannosidase constructs as shown in  FIGS. 5D ,  5 E,  6 D- 6 F.  FIGS. 5 and 6  show expression of constructs generated from the combinatorial DNA library which show significant mannosidase activity in  P. pastoris . Expression of pGC5 ( Saccharomyces  MNS1(m)/mouse mannosidase IB Δ99) ( FIGS. 5D and 6E ) produced a protein which has approximately 30% of all glycans trimmed to Man 5 GlcNAc 2 , while expression of pFB8 ( Saccharomyces  SEC12(m)/mouse mannosidase IA Δ187) ( FIG. 6F ) produced approximately 50% Man 5 GlcNAc 2  and expression of pBC18-5 ( Saccharomyces  VAN1(s)/ C. elegans  mannosidase IB Δ80) ( FIG. 5E ) produced 70% Man 5 GlcNAc 2 . 
     Example 6 
     Trimming In Vivo by α-1,2-Mannosidase 
     To ensure that the novel engineered strains of Example 4 in fact produced the desired Man 5 GlcNAc 2  structure in vivo, cell supernatants were tested for mannosidase activity (see  FIGS. 7-9 ). For each construct/host strain described below, HPLC was performed at 30° C. with a 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH 2  resin (5 μm) at a flow rate of 1.0 ml/min for 40 min. In  FIGS. 7 and 8 , degradation of the standard Man 9 GlcNAc 2  [b] was shown to occur resulting in a peak which correlates to Man 8 GlcNAc 2 . In  FIG. 7 , the Man 9 GlcNAc 2  [b] standard eluted at 24.61 min and Man 5 GlcNAc 2  [a] eluted at 18.59 min. In  FIG. 8 , Man 9 GlcNAc 2  eluted at 21.37 min and Man 5 GlcNAc 2  at 15.67 min. In  FIG. 9 , the standard Man 8 GlcNAc 2  [b] was shown to elute at 20.88 min. 
       P. pastoris  cells comprising plasmid pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in  FIG. 6F . The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity. A commercially available standard of 2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB) (Glyko, Novato, Calif.) was added to: BMMY ( FIG. 7A ), the supernatant from the above aliquot ( FIG. 7B ), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase from  Trichoderma reesei  (obtained from Contreras et al., WO 02/00856 A2) ( FIG. 7C ). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming. 
       P. pastoris  cells comprising plasmid pGC5 ( Saccharomyces  MNS1(m)/mouse mannosidase IB Δ99) were similarly grown and assayed. Cells were grown at room temperature in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in  FIG. 5D . The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity as shown in  FIG. 8B . A commercially available standard of Man9-2-AB (Glyko, Novato, Calif.) were added to: BMMY ( FIG. 8A ), supernatant from the above aliquot ( FIG. 8B ), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase from  Trichoderma reesei  (obtained from Contreras et al., WO 02/00856 A2) ( FIG. 8C ). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming. 
     Man9-2-AB was used as a substrate and it is evident that after 24 hours of incubation, mannosidase activity was virtually absent in the supernatant of the pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187) strain digest ( FIG. 7B ) and pGC5 ( Saccharomyces  MNS1(m)/mouse mannosidase IB Δ99) strain digest ( FIG. 8B ) whereas the positive control (purified α-1,2-mannosidase from  T. reesei  obtained from Contreras) leads to complete conversion of Man 9 GlcNAc 2  to Man 5 GlcNAc 2  under the same conditions, as shown in  FIGS. 7C and 8C . This is conclusive data showing in vivo mannosidase trimming in  P. pastoris  pGC5 strain; and pFB8 strain, which is distinctly different from what has been reported to date (Contreras et al., WO 02/00856 A2). 
       FIG. 9  further substantiates localization and activity of the mannosidase enzyme.  P. pastoris  comprising pBC18-5 ( Saccharomyces  VAN1(s)/ C. elegans  mannosidase IB Δ80) was grown at room temperature in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in  FIG. 5E . The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity as shown in  FIG. 9B . A commercially available standard of Man8-2-AB (Glyko, Novato, Calif.) was added to: BMMY ( FIG. 9A ), supernatant from the above aliquot pBC18-5 ( Saccharomyces  VAN1(s)/ C. elegans  mannosidase IB Δ80) ( FIG. 9B ), and BMMY containing media from a different fusion construct pDD28-3 ( Saccharomyces  MNN10(m) (from SwissProt 50108)/ H. sapiens  mannosidase IB Δ99) ( FIG. 9C ). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming.  FIG. 9B  demonstrates intracellular mannosidase activity in comparison to a fusion construct pDD28-3 ( Saccharomyces  MNN10(m)  H. sapiens  mannosidase IB Δ99) exhibiting a negative result ( FIG. 9C ). 
     Example 7 
     pH Optimum Assay of Engineered α-1,2-Mannosidase 
       P. pastoris  cells comprising plasmid pBB27-2 ( Saccharomyces  MNN10 (s) (from SwissProt 50108)/ C. elegans  mannosidase IB Δ31) were grown at room temperature in BMGY to an OD600 of about 17. About 80 μL of these cells were inoculated into 600 μL BMGY and were grown overnight. Subsequently, cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant (pH 6.43). The supernatant was removed for mannosidase pH optimum assays. Fluorescence-labeled Man 8 GlcNAc 2  (0.5 μg) was added to 20 μL of supernatant adjusted to various pH ( FIG. 11 ) and incubated for 8 hours at room temperature. Following incubation the sample was analyzed by HPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silica column (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 min and the column was maintained to 30° C. After eluting isocratically (68% A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60% B) was employed over 27 min to elute the glycans (18). Solvent A (acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. The column was equilibrated with solvent (68% A:32% B) for 20 min between runs. 
     Example 8 
     Engineering of  P. pastoris  to Produce N-Glycans with the Structure GlcNAcMan 5 GlcNAc 2    
     GlcNAc Transferase I activity is required for the maturation of complex and hybrid N-glycans (U.S. Pat. No. 5,834,251). Man 5 GlcNAc 2  may only be trimmed by mannosidase II, a necessary step in the formation of human glycoforms, after the addition of N-acetylglucosamine to the terminal α-1,3 mannose residue of the trimannose stem by GlcNAc Transferase I (Schachter, 1991 Glycobiology 1(5):453-461). Accordingly, a combinatorial DNA library was prepared including DNA fragments encoding suitably targeted catalytic domains of GlcNAc Transferase I genes from  C. elegans  and  Homo sapiens ; and localization sequences from GLS, MNS, SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2, MNN2, MNN5, YUR1, MNN1, and MNN6 from  S. cerevisiae  and  P. pastoris  putative α-1,2-mannosyltransferases based on the homology from  S. cerevisiae : D2, D9 and J3, which are KTR homologs. Table 10 includes but does not limit targeting peptide sequences such as SEC and OCH1, from  P. pastoris  and  K. lactis  GnTI, (See Table 6 and Table 10) 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 A representative combinatorial library of targeting peptide 
               
               
                 sequenes/catalytic domain for UDP-N-Acetylglucosaminyl Transferase I (GnTI) 
               
            
           
           
               
               
            
               
                   
                 Targeting peptide 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 OCHI(s) 
                 OCHI(m) 
                 OCHI(l) 
                 MNN9(s) 
                 MNN9(m) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Catalytic 
                 Human, GnTI, Δ38 
                 PB105 
                 PB106 
                 PB107 
                 PB104 
                 N/A 
               
               
                 Domain 
                 Human, GnTI, Δ86 
                 NB12 
                 NB13 
                 NB14 
                 NB15 
                 NB 
               
               
                   
                   C. elegans , GnTI, Δ88 
                 OA12 
                 OA13 
                 OA14 
                 OA15 
                 OA16 
               
               
                   
                   C. elegans , GnTI, Δ35 
                 PA12 
                 PA13 
                 PA14 
                 PA15 
                 PA16 
               
               
                   
                   C. elegans , GnTI, Δ63 
                 PB12 
                 PB13 
                 PB14 
                 PB15 
                 PB16 
               
               
                   
                   X. leavis , GnTI, Δ33 
                 QA12 
                 QA13 
                 QA14 
                 QA15 
                 QA16 
               
               
                   
                   X. leavis , GnTI, Δ103 
                 QB12 
                 QB13 
                 QB14 
                 QB15 
                 QB16 
               
               
                   
               
            
           
         
       
     
     Targeting peptide sequences were selected from OCH1 in  P. pastoris  (long, medium and short) (see Example 4) and MNN9 (SwissProt P39107) in  S. cerevisiae  (short and medium). Catalytic domains were selected from human GnTI with a 38 and 86 amino acid N-terminal deletion,  C. elegans  (gly-12) GnTI with a 35 and 63 amino acid deletion as well as  C. elegans  (gly-14) GnTI with a 88 amino acid N-terminal deletion and  X. leavis  GnTI with a 33 and 103 amino acid N-terminal deletion, respectively. 
     A portion of the gene encoding human N-acetylglucosaminyl Transferase I (MGATI, Accession# NM002406), lacking the first 154 bp, was amplified by PCR using oligonucleotides 5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ ID NO:72) and 5′-AGGTTAATTA AGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:73) and vector pHG4.5 (ATCC#79003) as template. The resulting PCR product was cloned into pCR2.1-TOPO and the correct sequence was confirmed. Following digestion with AscI and PacI the truncated GnTI was inserted into plasmid pJN1346 to create pNA. After digestion of pJN271 with NotI and AscI, the 120 bp insert was ligated into pNA to generate an in-frame fusion of the MNN9 transmembrane domain with the GnTI, creating pNA15. 
     The host organism is a strain of  P. pastoris  that is deficient in hypermannosylation (e.g. an och1 mutant), provides the substrate UDP-GlcNAc in the Golgi and/or ER (i.e., contains a functional UDP-GlcNAc transporter), and provides N-glycans of the structure Man 5 GlcNAc 2  in the Golgi and/or ER (e.g.  P. pastoris  pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187) from above). First,  P. pastoris  pFB8 was transformed with pPB 103 containing the  Kluyveromyces lactis  MNN2-2 gene (Genbank AN AF106080) (encoding UDP-GlcNAc transporter) cloned into BamHI and BglII site of pBLADE-SX plasmid (Cereghino et al. (2001)  Gene  263:159-169). Then the aforementioned combinatorial DNA library encoding a combination of exogenous or endogenous GnTI/localization genes was transformed and colonies were selected and analyzed for the presence of the GnTI construct by colony PCR. Our transformation and integration efficiency was generally above 80% and PCR screening can be omitted once robust transformation parameters have been established. 
     Protein Purification 
     K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Another screening method may be performed using a specific terminal GlcNAc binding antibody, or a lectin such as the GSII lectin from  Griffonia simplficolia , which binds terminal GlcNAc (EY Laboratories, San Mateo, Calif.). These screens can be automated by using lectins or antibodies that have been modified with fluorescent labels such as FITC or analyzed by MALDI-TOF. 
     Secreted K3 can be purified by Ni-affinity chromatography, quantified and equal amounts of protein can be bound to a high protein binding 96-well plate. After blocking with BSA, plates can be probed with a GSII-FACS lectin and screened for maximum fluorescent response. A preferred method of detecting the above glycosylated proteins involves the screening by MALDI-TOF mass spectrometry following the affinity purification of secreted K3 from the supernatant of 96-well cultured transformants. Transformed colonies were picked and grown to an OD600 of 10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested by centrifugation, washed in BMMY and resuspended in 250 ul of BMMY. Following 24 hours of induction, cells were removed by centrifugation, the supernatant was recovered and K3 was purified from the supernatant by Ni affinity chromatography. The N-glycans were released and analyzed by MALDI-TOF delayed extraction mass spectrometry as described herein. 
     In summary, the methods of the invention yield strains of  P. pastoris  that produce GlcNAcMan 5 GlcNAc 2  in high yield, as shown in  FIG. 10B . At least 60% of the N-glycans are GlcNAcMan 5 GlcNAc 2 . To date, no report exists that describes the formation of GlcNAcMan 5 GlcNAc 2  on secreted soluble glycoproteins in any yeast. Results presented herein show that addition of the UDP-GlcNAc transporter along with Gnu activity produces a predominant GlcNAcMan 5 GlcNAc 2  structure, which is confirmed by the peak at 1457 (m/z) ( FIG. 10B ). 
     Construction of Strain PBP-3: 
     The  P. pastoris  strain expressing K3, (Δoch1, arg-, ade-, his-) was transformed successively with the following vectors. First, pFB8 ( Saccharomyces  SEC12 (m)/mouse mannosidase IA Δ187) was transformed in the  P. pastoris  strain by electroporation. Second, pPB103 containing  Kluyveromyces lactis  MNN2-2 gene (Genbank AN AF106080) (encoding UDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al. (2001)  Gene  263:159-169) digested with BamHI and BelII enzymes was transformed in the  P. pastoris  strain. Third, pPB104 containing  Saccharomyces  MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacI fragment into pJN336 was transformed into the  P. pastoris  strain. 
     Example 9 
     Engineering  K. lactis  Cells to Produce N-Glycans with the Structure Man 5 GlcNAc 2    
     Identification and Disruption of the  K. lactis  OCH1 Gene 
     The OCH1 gene of the budding yeast  S. cerevisiae  encodes a 1,6-mannosyltransferase that is responsible for the first Golgi localized mannose addition to the Man 8 GlcNAc 2 N-glycan structure on secreted proteins (Nakanishi-Shindo et al. (1993)  J. Biol. Chem.;  268(35):26338-45). This mannose transfer is generally recognized as the key initial step in the fungal specific polymannosylation of N-glycan structures (Nakanishi-Shindo et al. (1993)  J. Biol. Chem.  268(35):26338-26345; Nakayama et al. (1992)  EMBO J.  11(7):2511-19; Morin-Ganet et al (2000)  Traffic  1(1):56-68). Deletion of this gene in  S. cerevisiae  results in a significantly shorter N-glycan structure that does not include this typical polymannosylation or a growth defect at elevated temperatures (Nakayama et al. (1992)  EMBO J.  11(7):2511-19). 
     The Och1p sequence from  S. cerevisiae  was aligned with known homologs from  Candida albicans  (Genbank accession # AAL49987), and  P. pastoris  along with the Hoc1 proteins of  S. cerevisiae  (Neiman et al (1997)  Genetics  145(3):637-45 and  K. lactis  (PENDANT EST database) which are related but distinct mannosyltransferases. Regions of high homology that were in common among Och1p homologs but distinct from the Hoc1p homologs were used to design pairs of degenerate primers that were directed against genomic DNA from the  K. lactis  strain MG1/2 (Bianchi et al (1987)  Current Genetics  12:185-192). PCR amplification with primers RCD33 (CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO:74) and RCD34 (CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:75) resulted in a 302 bp product that was cloned and sequenced and the predicted translation was shown to have a high degree of homology to Och1 proteins (&gt;55% to  S. cerevisiae  Och1p). 
     The 302 bp PCR product was used to probe a Southern blot of genomic DNA from  K. lactis  strain (MG1/2) with high stringency (Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Hybridization was observed in a pattern consistent with a single gene indicating that this 302 bp segment corresponds to a portion of the  K. lactis  genome and  K. lactis  (KlOCH1) contains a single copy of the gene. To clone the entire KlOCH1 gene, the Southern blot was used to map the genomic locus. Accordingly, a 5.2 kb BamHI/PstI fragment was cloned by digesting genomic DNA and ligating those fragments in the range of 5.2 kb into pUC 19 (New England Biolabs, Beverly, Mass.) to create a  K. lactis  subgenomic library. This subgenomic library was transformed into  E. coli  and several hundred clones were tested by colony PCR using RCD 33/34. The 5.2 kb clone containing the predicted KlOCH1 gene was sequenced and an open reading frame of 1362 bp encoding a predicted protein that is 46.5% identical to the  S. cerevisiae  OCH1 gene. The 5.2 kb sequence was used to make primers for construction of an och1::KAN R  deletion allele using a PCR overlap method (Davidson et al. (2002)  Microbiol.  148(Pt 8):2607-15). This deletion allele was transformed into two  K. lactis  strains and G418 resistant colonies selected. These colonies were screened by both PCR and for temperature sensitivity to obtain a strain deleted for the OCH1 ORF. The results of the experiment show strains which reveal a mutant PCR pattern, which were characterized by analysis of growth at various temperatures and N-glycan carbohydrate analysis of secreted and cell wall proteins following PNGase digestion. The och1 mutation conferred a temperature sensitivity which allowed strains to grow at 30° C. but not at 35° C.  FIG. 12A  shows a MALDI-TOF analysis of a wild type  K. lactis  strain producing N-glycans of Man 8 GlcNAc 2  [c] and higher. 
     Identification, Cloning, and Disruption of the  K. lactis  MNN1 Gene 
       S. cerevisiae  MNN1 is the structural gene for the Golgi α-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid type II membrane protein (Yip et al. (1994)  Proc Natl Acad Sci USA.  91(7):2723-7). Both N-linked and O-linked oligosaccharides isolated from mnn1 mutants lack α-1,3-mannose linkages (Raschke et al. (1973)  J Biol Chem.  248(13):4660-66). 
     The Mnn1p sequence from  S. cerevisiae  was used to search the  K. lactis  translated genomic sequences (PEDANT). One 405 bp DNA sequence encoding a putative protein fragment of significant similarity to Mnn1p was identified. An internal segment of this sequence was subsequently PCR amplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ ID NO:76) and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:77) and used to probe a Southern blot of genomic DNA from  K. lactis  strain (MG1/2). Based on the Southern hybridization data a 4.2 Kb BamHI-PstI fragment was cloned by generating a size-selected library as described herein. A single clone containing the  K. lactis MNN 1 gene was identified by whole colony PCR using primers KMN1 and KMN2 and sequenced. Within this clone a 2241 bp ORF was identified encoding a predicted protein that was 34% identical to the  S. cerevisiae  MNN1 gene. Primers were designed for construction of a mnn1::NAT deletion allele using the PCR overlap method (Davidson et al. (2002)  Microbiol.  148(Pt 8):2607-15). 
     This disruption allele was transformed into a strain of  K. lactis  by electroporation and nourseothricin resistant transformants were selected and PCR amplified for homologous insertion of the disruption allele. Strains that reveal a mutant PCR pattern may be subjected to N-glycan carbohydrate analysis of a known reporter gene. 
       FIG. 12B  depicts the N-glycans from the  K. lactis  och1 mnn1 deletion strain observed following PNGase digestion the MALDI-TOF as described herein. The predominant peak at 1908 (m/z) indicated as [d] is consistent with the mass of Man 9 GlcNAc 2 . 
     Additional methods and reagents which can be used in the methods for modifying the glycosylation are described in the literature, such as U.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No. 6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat. No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S. Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854, U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No. 5,047,335. Appropriate yeast expression systems can be obtained from sources such as the American Type Culture Collection, Rockville, Md. Vectors are commercially available from a variety of sources. 
     Example 10 
     Identification, Cloning and Deletion of the ALG3 Gene in  P. pastoris  and  K. lactis    
     Degenerate primers were generated based on an alignment of Alg3 protein sequences from  S. cerevisiae, H. sapiens , and  D. melanogaster  and were used to amplify an 83 bp product from  P. pastoris  genomic DNA: 5′-GGTGTTTTGTTTTCTAGATCTTTGCAYTAYCARTT-3′ (SEQ ID NO:78) and 5′-AGAATTTGGTGGGTAAGAAITCCARCACCAYTCRTG-3′ (SEQ ID NO:79). The resulting PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and sequence analysis revealed homology to known ALG3/RHK1/NOT56 homologs (Genbank NC — 001134.2, AF309689, NC — 003424.1). Subsequently, 1929 bp upstream and 2738 bp downstream of the initial PCR product were amplified from a  P. pastoris  genomic DNA library (Boehm (1999) Yeast 15(7):563-72) using the internal oligonucleotides 5′-CCTAAGCTGGTATGCGTTCTCTTTGCCATATC-3′ (SEQ ID NO:80) and 5′-GCGGCATAAACAATAATAGATGCTATAAAG-3′ (SEQ ID NO:81) along with T3 (5′-AATTAACCCTCACTAAAGGG-3′) (SEQ ID NO:49) and T7 (5′-GTAA TACGACTCACTATAGGGC-3′) (SEQ ID NO:48) (Integrated DNA Technologies, Coralville, Iowa) in the backbone of the library bearing plasmid lambda ZAP II (Stratagene, La Jolla, Calif.). The resulting fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. From this sequence, a 1395 bp ORF was identified that encodes a protein with 35% identity and 53% similarity to the  S. cerevisiae  ALG3 gene (using BLAST programs). The gene was named PpALG3. 
     The sequence of PpALG3 was used to create a set of primers to generate a deletion construct of the PpALG3 gene by PCR overlap (Davidson et al (2002)  Microbiol.  148(Pt 8):2607-15). Primers below were used to amplify 1 kb regions 5′ and 3′ of the PpALG3 ORF and the KAN R  gene, respectively: 
                            RCD142           (SEQ ID NO: 82)           (5′-CCACATCATCCGTGCTACATATAG-3′),                       RCD144           (SEQ ID NO: 83)           (5′-ACGAGGCAAGCTAAACAGATCTCGAAGTATCGAGGGTTATC                       CAG-3′),                       RCD145           (SEQ ID NO: 84)           (5′-CCATCCAGTGTCGAAAACGAGCCAATGGTTCATGTCTATAA                       ATC-3′),                       RCD147           (SEQ ID NO: 85)           (5′-AGCCTCAGCGCCAACAAGCGATGG-3′),                       RCD143           (SEQ ID NO: 86)           (5′-CTGGATAACCCTCGATACTTCGAGATCTGTTTAGCTTGCCT                       CGT-3′),           and                       RCD146           (SEQ ID NO: 87)           (5′-GATTTATAGACATGAACCATTGGCTCGTTTTCGACACTGGA                       TGG-3′).            
Subsequently, primers RCD 142 and RCD 147 were used to overlap the three resulting PCR products into a single 3.6 kb alg3::KAN R  deletion allele.
 
Identification, Cloning and Deletion of the ALG3 Gene in  K. lactis.  
 
     The ALG3p sequences from  S. cerevisiae, Drosophila melanogaster, Homo sapiens  etc were aligned with  K. lactis  sequences (PENDANT EST database). Regions of high homology that were in common homologs but distinct in exact sequence from the homologs were used to create pairs of degenerate primers that were directed against genomic DNA from the  K. lactis  strain MG1/2 (Bianchi et al, 1987). In the case of ALG3, PCR amplification with primers 
                            KAL-1           (SEQ ID NO: 88)           (5′-ATCCTTTACCGATGCTGTAT-3′)           and                       KAL-2           (SEQ ID NO: 89)           (5′-ATAACAGTATGTGTTACACGCGTGTAG-3′)            
resulted in a product that was cloned and sequenced and the predicted translation was shown to have a high degree of homology to Alg3p proteins (&gt;50% to  S. cerevisiae  Alg3p).
 
     The PCR product was used to probe a Southern blot of genomic DNA from  K. lactis  strain (MG1/2) with high stringency (Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Hybridization was observed in a pattern consistent with a single gene. This Southern blot was used to map the genomic loci. Genomic fragments were cloned by digesting genomic DNA and ligating those fragments in the appropriate size-range into pUC19 to create a  K. lactis  subgenomic library. This subgenomic library was transformed into  E. coli  and several hundred clones were tested by colony PCR, using primers KAL-1 and KAL-2. The clones containing the predicted KlALG3 and KlALG61 genes were sequenced and open reading frames identified. 
     Primers for construction of an alg3::NAT R  deletion allele, using a PCR overlap method (Davidson et al. (2002)  Microbiol.  148(Pt 8):2607-15), were designed and the resulting deletion allele was transformed into two  K. lactis  strains and NAT-resistant colonies selected. These colonies were screened by PCR and transformants were obtained in which the ALG3 ORF was replaced with the och1::NAT R  mutant allele. 
     Example 11 
     Generation of an Alg3 Och1 Mutant Strain Expressing an α-1,2-Mannosidase, GnT1 and GnTII for Production of a Human-Like Glycoprotein 
     A  P. pastoris  alg3::KAN R  deletion construct was generated as described in Example 10. Approximately 5 μg of the resulting PCR product was transformed into strain PBP-3 (see Example 3), and colonies were selected on YPD medium containing 200 μg/ml G418. One strain out of 20 screened by PCR was confirmed to contain the correct integration of the alg3::KAN R  mutant allele and lack the wild-type allele. This strain was named RDP27 ( FIG. 36 ). 
     A library of GnTII constructs was then generated, which was comprised of in-frame fusions of the leader library with the catalytic domains of GnTII genes from human and rat sources (WO 02/00879). This library was created in a  P. pastoris  integration vector containing the NST R  gene conferring resistance to the drug nourseothricin. The library plasmids were linearized with EcoRI, transformed into strain RDP27 by electroporation, and the resulting strains were screened by analysis of the released glycans from purified K3. A  P. pastoris  strain expressing the rat GnTII fused in-frame to the  S. cerevisiae  MNN9 (s) construct was named PBP6-5 ( FIG. 36 ). 
     Generation of GnTII Expression Constructs 
     The construction of a GnTI expression vector (pNA15) containing a human GnTI gene fused with the N-terminal part of  S. cerevisiae  MNN9 gene is described in Choi et al. (2003)  Proc Natl Acad Sci USA.  100(9):5022-27. In a similar fashion, the rat GnTII gene was cloned. The rat GnTII gene (GenBank accession number U21662) was PCR amplified using Takara EX Taq™ polymerase (Panvera) from rat liver cDNA library (Clontech) with RAT1 (5′-TTCCTCACTGCAGTCTTCTATAACT-3′) (SEQ ID NO:90) and RAT2 (5′-TGGAGACCATGAGGTTCCGCATCTAC-3′) (SEQ ID NO:91) primers. The PCR product was then cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced. Using this vector as a template, the AscI-PacI fragment of GnTII, encoding amino-acids 88-443, was amplified with Pfu Turbo polymerase (Stratagene) and primers, 
                            RAT44           (SEQ ID NO: 92)           (5′-TT GGCGCGCC TCCCTAGTGTACCAGTTGAACTTTG-3′)           and                       RAT11           (SEQ ID NO: 93)           (5′-GA TTAATTAA CTCACTGCAGTCTTCTATAACT-3′)            
respectively, introduced AscI and PacI restriction sites are underlined). Following confirmation by sequencing, the catalytic domain of rat GnTII was than cloned downstream of the PMA1 promoter as a AscI-PacI fragment in pBP124. In the final step, the gene fragment encoding the  S. cerevisiae  Mnn2 localization signal was cloned from pJN281 as a NotI-AscI fragment to generate an in-frame fusion with the catalytic domain of GnTII, to generate plasmid pTC53.
 
     Example 12 
     Cloning and Expression of GnTIII to Produce Bisecting GlcNAcs which Boost Antibody Functionality 
     The addition of an N-acetylglucosamine to the GlcNAc 2 Man 3 GlcNAc 2  structure by N-acetylglucosaminyltransferases III yields a so-called bisected N-glycan (see  FIG. 15 ). This structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC) (Umana et al. (1999)  Nat. Biotechnol.  17(2):176-80). 
     A host cell such as a yeast strain capable of producing glycoproteins with bisected N-glycans is engineered according to the invention, by introducing into the host cell a GnTIII activity. Preferably, the host cell is transformed with a nucleic acid that encodes GnTIII (e.g., a mammalian such as the murine GnTIII shown in  FIG. 24 ) or a domain thereof having enzymatic activity, optionally fused to a heterologous cell signal targeting peptide (e.g., using the libraries and associated methods of the invention.) 
     IgGs consist of two heavy-chains (V H , C H 1, C H 2 and C H 3 in  FIG. 22 ), interconnected in the hinge region through three disulfide bridges, and two light chains (V L , C L  in  FIG. 22 ). The light chains (domains V L  and C L ) are linked by another disulfide bridge to the C H 1 portion of the heavy chain and together with the C H 1 and V H  fragment make up the so-called Fab region. Antigens bind to the terminal portion of the Fab region. The Fc region of IgGs consists of the C H 3, the C H 2 and the hinge region and is responsible for the exertion of so-called effector functions (see below). 
     The primary function of antibodies is binding to an antigen. However, unless binding to the antigen directly inactivates the antigen (such as in the case of bacterial toxins), mere binding is meaningless unless so-called effector-functions are triggered. Antibodies of the IgG subclass exert two major effector-functions: the activation of the complement system and induction of phagocytosis. The complement system consists of a complex group of serum proteins involved in controlling inflammatory events, in the activation of phagocytes and in the lytical destruction of cell membranes. Complement activation starts with binding of the C1 complex to the Fc portion of two IgGs in close proximity. C1 consists of one molecule, C1q, and two molecules, C1r and C1s. Phagocytosis is initiated through an interaction between the IgG&#39;s Fc fragment and Fc-gamma-receptors (FcγRI, II and III in  FIG. 22 ). Fc receptors are primarily expressed on the surface of effector cells of the immune system, in particular macrophages, monocytes, myeloid cells and dendritic cells. 
     The C H 2 portion harbors a conserved N-glycosylation site at asparagine 297 (Asn297). The Asn297 N-glycans are highly heterogeneous and are known to affect Fc receptor binding and complement activation. Only a minority (i.e., about 15-20%) of IgGs bears a disialylated, and 3-10% have a monosialylated N-glycan (reviewed in Jefferis (2001)  Biopharm.  14:19-26). Interestingly, the minimal N-glycan structure shown to be necessary for fully functional antibodies capable of complement activation and Fc receptor binding is a pentasacharide with terminal N-acetylglucosamine residues (GlcNAc 2 Man 3 ) (reviewed in Jefferis, R., Glycosylation of human IgG Antibodies. BioPharm, 2001). Antibodies with less than a GlcNAc 2 Man 3  N-glycan or no N-glycosylation at Asn297 might still be able to bind an antigen but most likely will not activate the crucial downstream events such as phagocytosis and complement activation. In addition, antibodies with fungal-type N-glycans attached to Asn297 will in all likelihood solicit an immune-response in a mammalian organism which will render that antibody useless as a therapeutic glycoprotein. 
     Cloning and Expression of GnTIII 
     The DNA fragment encoding part of the mouse GnTIII protein lacking the TM domain is PCR amplified from murine (or other mammalian) genomic DNA using 
     forward (5′-TCCTGGCGCGCCTTCCCGAGAGAACTGGCCTCCCTC-3′) (SEQ ID NO:94) and
 
reversed (5′-AATTAATTAACCCTAGCCCTCCGCTGTATCCAACTTG-3′) (SEQ ID NO:95) primers. Those primers include AscI and PacI restriction sites that may be used for cloning into the vector suitable for the fusion with leader library.
 
     The nucleic acid (SEQ ID NO:45) and amino acid (SEQ ID NO:46) sequences of murine GnTIII are shown in  FIG. 24 . 
     Cloning of Immunoglobulin-Encoding Sequences 
     Protocols for the cloning of the variable regions of antibodies, including primer sequences, have been published previously. Sources of antibodies and encoding genes can be, among others, in vitro immunized human B cells (see, e.g., Borreback et al. (1988)  Proc. Natl. Acad. Sci. USA  85:3995-3999), peripheral blood lymphocytes or single human B cells (see, e.g., Lagerkvist et al. (1995)  Biotechniques  18:862-869; and Terness et al. (1997)  Hum. Immunol.  56:17-27) and transgenic mice containing human immunoglobulin loci, allowing the creation of hybridoma cell-lines. 
     Using standard recombinant DNA techniques, antibody-encoding nucleic acid sequences can be cloned. Sources for the genetic information encoding immunoglobulins of interest are typically total RNA preparations from cells of interest, such as blood lymphocytes or hybridoma cell lines. For example, by employing a PCR based protocol with specific primers, variable regions can be cloned via reverse transcription initiated from a sequence-specific primer hybridizing to the IgG C H 1 domain site and a second primer encoding amino acids 111-118 of the murine kappa □ constant region. The V H  and V K  encoding cDNAs can then be amplified as previously published (see, e.g., Graziano et al. (1995)  J Immunol.  155(10):4996-5002; Welschof et al. (1995)  J. Immunol. Methods  179:203-214; and Orlandi et al. (1988)  Proc. Natl. Acad. Sci. USA  86:3833). Cloning procedures for whole immunoglobulins (heavy and light chains) have also been published (see, e.g., Buckel et al. (1987)  Gene  51:13-19; Recinos et al. (1994)  Gene  149: 385-386; Recinos et al. (1995)  Gene  158:311-12). Additional protocols for the cloning and generation of antibody fragment and antibody expression constructs have been described in Antibody Engineering, Kontermann and Dübel (2001), Eds., Springer Verlag: Berlin Heidelberg New York. 
     Fungal expression plasmids encoding heavy and light chain of immunoglobulins have been described (see, e.g., Abdel-Salam et al. (2001)  Appl. Microbiol. Biotechnol.  56:157-164; and Ogunjimi et al. (1999)  Biotechnology Letters  21:561-567). One can thus generate expression plasmids harboring the constant regions of immunoglobulins. To facilitate the cloning of variable regions into these expression vectors, suitable restriction sites can be placed in close proximity to the termini of the variable regions. The constant regions can be constructed in such a way that the variable regions can be easily in-frame fused to them by a simple restriction-digest/ligation experiment.  FIG. 23  shows a schematic overview of such an expression construct, designed in a very modular way, allowing easy exchange of promoters, transcriptional terminators, integration targeting domains and even selection markers. 
     As shown in  FIG. 23 , V L  as well as V H  domains of choice can be easily cloned in-frame with C L  and the C H  regions, respectively. Initial integration is targeted to the  P. pastoris  AOX locus (or homologous locus in another fungal cell) and the methanol-inducible AOX promoter will drive expression. Alternatively, any other desired constitutive or inducible promoter cassette may be used. Thus, if desired, the 5′AOX and 3′AOX regions as well as transcriptional terminator (TT) fragments can be easily replaced with different TT, promoter and integration targeting domains to optimize expression. Initially the alpha-factor secretion signal with the standard KEX protease site is employed to facilitate secretion of heavy and light chains. The properties of the expression vector may be further refined using standard techniques. 
     An Ig expression vector such as the one described above is introduced into a host cell of the invention that expresses GnTIII, preferably in the Golgi apparatus of the host cell. The Ig molecules expressed in such a host cell comprise N-glycans having bisecting GlcNAcs. 
     Example 13 
     Generation of Yeast Strain YSH-1 (Δoch1, α1,2-Mannosidase, GnTI) 
     The previously reported  P. pastoris  strain BK64 (Choi et al. (2003)  Proc Natl Acad Sci USA.  100(9):5022-7), a triple auxotroph (ADE, ARG, HIS) possessing the OCH1 knock-out and expressing the kringle 3 domain (K3) of human plasminogen, was used as the host strain. BK64 was transformed with the plasmid pPB 103 linearized with the restriction enzyme EcoNI to introduce the  K. lactis  UDP-N-acetylglucosamine transporter into the host cell, thus creating the strain PBP-1. The mouse MnsI was introduced into this strain by transformation with the plasmid pFB8 linearized with the restriction enzyme EcoNI, generating strain PBP-2. K3 glycan analysis from proteins isolated from strain PBP-2 demonstrated that the primary glycoform present was Man 5 GlcNAc 2 . 
     PBP-2 was subsequently transformed with the human GnTI plasmid pNA15 linearized with the restriction enzyme AatII, generating the strain PBP-3. Analysis of the K3 glycoforms produced in strain PBP-3 demonstrated that the hybrid glycan GlcNAcMan 5 GlcNAc 2  was the predominant structure. To recover the URA3 marker from PBP-3, this strain was grown in YPD prior to selection on minimal media containing 5-Fluoroorotic (5-FOA, BioVectra) and uracil (Boeke et al. (1984)  Mol. Gen. Genet.  197:345-346). The recovered Ura-minus strain producing GlcNAcMan 5 GlcNAc 2  glycoforms was designated YSH-1 ( FIG. 36 ). The N-glycan profile from strain YSH-1 is shown in  FIG. 25  (top) and displays a predominant peak at 1465 m/z corresponding to the mass of GlcNAcMan 5 GlcNAc 2  [d]. 
     Example 14 
     Generation of Yeast Strain YSH-37 ( P. pastoris  Expressing Mannosidase II) 
     YSH-1 (Example 13) was transformed with the  D. melanogaster  mannosidase IIΔ74/ S. cerevisiae  MNN2(s) plasmid (pKD53) linearized with the restriction enzyme ApaI, generating strain YSH-37 ( FIG. 36 ). Analysis of the K3 glycan structures produced in strain YSH-37 ( FIG. 25  (bottom)) demonstrated that the predominant glycoform at 1140 m/z corresponds to the mass of GlcNAcMan 3 GlcNAc 2  [b] and other glycoforms GlcNAcMan 4 GlcNAc 2  [c] at 1303 m/z and GlcNAcMan 5 GlcNAc 2  [d] at 1465 m/z. 
     Example 15 
     Generation of Yeast Strain YSH-44 
     Strain YSH-37 (Example 14) was transformed with a plasmid encoding a rat GnTII/MNN2 (s) leader, pTC53, linearized with the restriction enzyme EcoRI. The resulting strain, YSH-44 ( FIG. 36 ), produced a K3 N-glycan having a single glycoform at 1356 m/z, corresponding to the mass of GlcNAc 2 Man 3 GlcNAc 2  [x], by positive mode MALDI-TOF mass spectrometry ( FIG. 29 ). 
     β-N-acetylhexosaminidase Digestion 
     The glycans from YSH-44 were released and separated from the glycoproteins by a modification of a previously reported method (Papac, et al. A. J. S. (1998)  Glycobiology  8, 445-454). After the proteins were reduced and carboxymethylated and the membranes blocked, the wells were washed three time with water. The protein was deglycosylated by the addition of 30 μl of 10 mM NH 4 HCO 3  pH 8.3 containing one milliunit of N-glycanase (Glyko, Novato, Calif.). After a 16 hr digestion at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness. The glycans were then dried in aSC210A speed vac (Thermo Savant, Halbrook, N.Y.). The dried glycans were put in 50 mM NH 4 Ac pH 5.0 at 37° C. overnight and 1 mU of hexos (Glyko, Novato, Calif.) was added. 
     Example 16 
     Construction of Plasmid pJN 348 
     The plasmid pBLURA-SX (from Jim Cregg) was digested with BamHI and BglII to release the AOX expression cassette. The BamHI fragment containing the GAPDH/CYC1 expression cassette from pJN261 ( FIG. 4B ) (Example 4) was then ligated into the pBLURA-SX backbone to create pJN338. The plasmid pJN338 was cut with NotI and PacI and the two oligonucleotides 
     5′-GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT-3′ (SEQ ID NO:96) and 
     5′-TAAGGCGCGCC GAATTCATTTAAATCTGCAGGGC-3′ (SEQ ID NO:97) that had been annealed in vitro, were ligated into the open sites, to create pJN348. 
     Example 17 
     Construction of an Integration Plasmid pRCD259 
     The PpURA3 containing GAPDH expression vector pJN348 was linearized with XhoI and blunted with T4 DNA polymerase and calf intestinal phosphatase (CIP) treated. The HYG resistance marker was digested from pAG32 with BglII and SacI and blunted, then ligated into pJN348 to create pRCD259 which can be used as a HYG expression vector that integrates at the PpURA3 locus. 
     Example 18 
     Generation of GnTIII Fusion Constructs 
     Fusion constructs between mammalian GnTIII and yeast targeting sequences were made using mouse Mgat3 gene (GenBank accession number L39373, Bhaumik et al., 1995). Three DNA fragments corresponding to N-terminal deletions Δ32, Δ86, and Δ212 of the mouse GnTIII gene were PCR amplified using Pfu Turbo polymerase (Stratagene) with forward 
     MG3-B (5′-TCCTGGCGCGCCTTCCCGAGAGAACTGGCCTCCCTC-3′) (SEQ ID NO:98), 
     MG3-C (5′-CCGAGGCGCGCCACAGAGGAACTGCACCGGGTG-3′) (SEQ ID NO:99), 
     MG3-D (5′-ACCGAGGCGCGCCATCAACGCCATCAACATCAACCAC-3′) (SEQ ID NO:100), 
     and reverse
 
MG3-A (5′-AATTAATTAACCCTAGCCCTCCGCTGTATCCAACTTG-3′) (SEQ ID NO:101) primers. The PCR products were then cloned into pJN 348 vector as AscI-PacI fragments and sequenced. The resulting vectors pVA (GnTIII Δ32), pVB (GnTIII Δ86), and pVC (GnTIII Δ212) were digested with NotI-AscI enzymes and used for the ligation with yeast leader library (leaders 20-67). These targeting peptides are fused to the catalytic domains selected from the mouse GnTIII with 32, 86, 212 amino acid N-terminal deletions. For example, the MNN2 targeting peptide from  S. cerevisiae  (long, medium and short) and GNT1 from  K. lactis  (short, and medium) (see Example 11) are shown in Table 11.
 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 A representative combinatorial library of targeting 
               
               
                 peptide sequences/catalytic domains exhibiting 
               
               
                 UDP-N-Acetylglucosaminyltransferase III (GnTIII) 
               
               
                 activity in  P. pastoris  YSH-1 
               
            
           
           
               
               
            
               
                   
                 Targeting peptide 
               
            
           
           
               
               
               
               
               
            
               
                   
                 
                   S. 
                 
                 
                   S. 
                 
                 
                   S. 
                 
                 
                   K. 
                 
               
               
                   
                 
                   cerevisiae 
                 
                 
                   cerevisiae 
                 
                 
                   cerevisiae 
                 
                 
                   lactis 
                 
               
               
                   
                 MNN2(s) 
                 MNN2(m) 
                 MNN2(l) 
                 GNT1(m) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Catalytic 
                 Mouse 
                 50% 
                 30-40% 
                 20-30% 
                 0% 
               
               
                 Domain 
                 GnTIII 
                 (pVA53) 
                 (pVA54) 
                 (pVA55) 
                 (pVA51) 
               
               
                   
                 Δ32 
               
               
                   
                 Mouse 
                 20-30%   
                 30-40% 
                 20-30% 
                 0% 
               
               
                   
                 GnTIII 
                 (pVB53) 
                 (pVB54) 
                 (pVB55) 
                 (pVB51) 
               
               
                   
                 Δ86 
               
               
                   
                 Mouse 
                  0% 
                    0% 
                    0% 
                 0% 
               
               
                   
                 GnTIII 
                 (pVC53) 
                 (pVC54) 
                 (pVC55) 
                 (pVC51) 
               
               
                   
                 Δ212 
               
               
                   
               
            
           
         
       
     
     Example 19 
     Engineering of  P. pastoris  to Produce Bisected GlcNAc 2 Man 5 GlcNAc 2    
     The  P. pastoris  strain producing GlcNAcMan 5 GlcNAc 2  (PBP-3) (see Example 8) was counterselected on 5-FOA, thereby selecting for loss of the URA3+ marker and a ura3-phenotype. This strain, designated YSH-1 ( FIG. 36 ), was transformed with the library of N-acetylglucosaminyltransferase III (GnTIII) catalytic domains (vectors pVA, pVB, and pVC) and leaders. Transformants were grown at 30° C. in BMGY to an OD600 of about 10, harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion (Example 3). The N-glycans were analyzed with a MALDI-TOF MS (Example 3). The GnTIII activities are shown in Table 11. The number of (+)s, as used herein, indicates the relative levels of bisected N-glycan production of % neutral glycans. Targeting peptide sequences were selected from selected from the group consisting of:  Saccharomyces  GLS 1,  Saccharomyces  MNS 1,  Saccharomyces  SEC 12,  Pichia  SEC,  Pichia  OCH1,  Saccharomyces  MNN9,  Saccharomyces  VAN1,  Saccharomyces  ANP1,  Saccharomyces  HOC1,  Saccharomyces  MNN10,  Saccharomyces  MNN11,  Saccharomyces  MNT1,  Pichia  D2,  Pichia  D9,  Pichia  J3,  Saccharomyces  KTR1,  Saccharomyces  KTR2,  Kluyveromyces  GnTI,  Saccharomyces  MNN2,  Saccharomyces  MNN5,  Saccharomyces  YUR1,  Saccharomyces  MNN1, and  Saccharomyces  MNN6. The pVA53 transformants exhibiting the bisecting GlcNAc (e.g. GlcNAc 2 Man 5 GlcNAc 2 ) were designated PBP26 ( FIG. 36 ). 
     Example 20 
     Engineering of  P. pastoris  YSH-44 to Produce Bisected GlcNAc 3 Man 3 GlcNAc 2    
     For the expression of GnTIII in the strain YSH-44 ( FIG. 36 ), GnTIII constructs from vectors pVA53, pVB53, pVA54, and pVB54 were transferred as NotI-PacI fragments into pRCD259 to generate vectors pPB 135, pPB 137, pPB 136, and pPB138. The vectors contain HYG resistance marker and  P. pastoris  URA3 gene as targeting sequence for genomic integration. Plasmids are linearized with SalI, transformed into strain YSH-44 by electroporation, selected on medium containing hygromycin and the resulting strains are screened by analysis of the released glycans from purified K3. Transformants were grown at 24° C. in BMGY to an OD600 of about 10, harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot (Example 3). The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion. The N-glycans were analyzed with a MALDI-TOF MS (Example 3). The pPB 135 transformants exhibiting the bisecting GlcNAc (e.g. GlcNAc 2 Man 5 GlcNAc 2 ) were designated YSH-57 ( FIG. 36 ). Table 11 depicts the activity of the mouse GnTIII. 
     Example 21 
     Engineering of  P. pastoris  PBP6-5 to Produce Bisected GlcNAc 3 Man 3 GlcNAc 2    
     The  P. pastoris  PBP6-5 (Example 11) was transformed with the plasmid pPB 135 (Table 11) encoding a mouse GnTIII catalytic domain (Δ32) ligated in frame to a targeting peptide derived from  S. cerevisiae  MNN2. Transformants were grown at 30° C. in BMGY to an OD600 of about 10, harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion (Example 3). The N-glycans were analyzed with a MALDI-TOF MS (Example 3). Transformants exhibiting the bisecting GlcNAc (e.g. GlcNAc 2 Man 3 GlcNAc 2 ) were designated PBP-38 ( FIG. 36 ). Table 11 depicts the activity of the mouse GnTIII. 
     Example 22 
     In Vitro GnTIII Activity Assay Using Substrate GlcNAcMan 5 GlcNAc 2  in Engineered  P. pastoris  Strain YSH-57 
     To test any potential ex vivo GnTIII activity in the  P. pastoris  strain, YSH-57 cell culture supernatants were tested for GnTIII activity.  P. pastoris  YSH-57 cells were grown at 24° C. in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for GnTIII assays and the remainder was used for the recovery of secreted soluble K3. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion (Example 3). The earlier removed aliquot of the supernatant was further tested for the presence of secreted GnTIII activity. GlcNAcMan 5 GlcNAc 2  purified from K3 expressed in PBP-3 strain was added to: BMMY (A) 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) in BMMY (B); the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C); the supernatant of YSH-57+1 mM UDP-GlcNAc (D). After incubation for 8 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of GnTIII activity. 
     Example 23 
     In Vitro GnTIII Activity Assay Substrate GlcNAc 2 Man 3 GlcNAc 2  in Engineered  P. pastoris  Strain YSH-57 
     To test any potential ex vivo GnTIII activity in the  P. pastoris  strain YSH-57 cell culture supernatants were tested for GnTIII activity.  P. pastoris  YSH-57 cells were grown at 24° C. in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for GnTIII assays and the remainder was used for the recovery of secreted soluble K3. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion (Example 3). The earlier removed aliquot of the supernatant was further tested for the presence of secreted GnTIII activity. GlcNAc 2 Man 3 GlcNAc 2  purified from K3 expressed in YSH-44 strain was added to: BMMY (A) 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) in BMMY (B); the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C). After incubation for 8 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of GnTIII activity. 
     Example 24 
     Cloning and Expression of GnTIV in  P. pastoris    
     The DNA fragment encoding part of the human GnTIV protein isoenzyme A (MGAT4A) lacking the TM domain was PCR amplified from human cDNA using 
     forward HGIV-2 (5′-CTGATTGCTTATCAACGAGAATTCCTTG-3′) (SEQ ID NO:102) and
 
reversed HGIV-3 (5′-TGTTGGTTTCTCAGATGATCAGTTGGTG-3′) primers (SEQ ID NO:103), cloned into the pCR2.1-TOPO vector (Invitrogen), and sequenced. For cloning into a vector suitable for the fusion with a leader library, DNA fragments were PCR amplified with primers that include AscI and Pad restriction sites. AscI forward primers: HGIV-ASC1 (5′-TGGGCGCGCCAAACTGATTGCTTATCAACGAGAA-3′) (SEQ ID NO:104), HGIV-ASC2 (5′-AGTGGGCGCGCCTTGAATAAGTTTTCAGATAATACC-3′) (SEQ ID NO:105), HGIV-ASC3 (5′-AAGGGCGCGCCCAAGTGCCAAGTATTTATTATC-3′) (SEQ ID NO:106). PacI reverse primer HGIV-PAC (5′-GTTTAATTAAGATCAGTTGGTGGCTTTTTTAATATG-3′) (SEQ ID NO:107). The nucleic acid and amino acid sequences of the human GnTIV A are shown in  FIG. 41 .
 
     Similarly, the DNA fragment encoding part of the human GnTIV protein isoenzyme B (MGAT4B) lacking the TM domain was PCR amplified from human cDNA using forward HGIVB-2 (5′-AGCGGCCAGAAAGGCGACGTTGTGGAC-3′) (SEQ ID NO:108) and reverse HGIVB-3 (5′-TACCCTCAGAAGCCCGCAGCTTAGTC-3′) (SEQ ID NO:109) primers, cloned into the pCR2.1-TOPO vector (Invitrogen), and sequenced. For cloning into a vector suitable for the fusion with a leader library, DNA fragments were PCR amplified with primers that include AscI and PacI restriction sites. AscI forward primers: HGIVB-A 1 (5′-AGCGGGCGCGCCGGCGACGTTGTGGACGTTTAC-3′) (SEQ ID NO:110), HGIVB-A2 (5′-CCGTGGCGCGCCTCACACCGGCACGTGCTGCAC-3′) (SEQ ID NO:111). Pad′ reverse primer HGIVB-P (5′-TGTTAATTAAGCTTAGTCGGCCTTTTTCAGGAAG-3′) (SEQ ID NO:112). The nucleic acid (SEQ ID NO:40) and amino acid (SEQ ID NO:39) sequences of human GnTIV B are shown in  FIG. 42 . 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Plasmids containing GnTV or GnTIV Fusion Constructs 
               
               
                 For Expression of Multiantennary Structures 
               
            
           
           
               
               
               
            
               
                   
                 leader 
                   
               
            
           
           
               
               
               
            
               
                 Catalytic domain 
                 HYG vector 
                 KAN vector 
               
               
                   
               
               
                 Mouse GnT V 
                   
                   
               
               
                  Δ95-53 
                   
                 pPB146 
               
               
                 Δ145-53 
                 pPB125 
                 pPB140 
               
               
                 Δ145-54 
                 pPB130 
               
               
                 Δ209-53 
                 pPB126 
               
               
                 Δ209-54 
                 pPB131 
               
               
                 Human GnT IVA 
               
               
                  Δ32-53 
                 pPB127 
               
               
                  Δ82-53 
                 pPB128 
                 pPB141 
               
               
                 Δ103-53 
                 pPB129 
               
               
                  Δ32-54 
                 pPB132 
               
               
                  Δ82-54 
                 pPB133 
               
               
                 Δ103-54 
                 pPB134 
               
               
                 Human GnT IVB 
               
               
                  Δ32-53 
                 pPB143 
               
               
                 Δ104-53 
                 pPB144 
               
               
                 Human GnT IX 
               
               
                  Δ43-53 
                   
                 pPB176 
               
               
                   
               
            
           
         
       
     
     Example 25 
       P. pastoris  Strain Producing Triantennary Glycan Structures 
       P. pastoris  YSH-44 strain (Example 15) producing complex glycan structures was transformed with the plasmid pPB144 (Table 12) containing a gene fragment encoding the human GnTIVB catalytic domain (Δ104) ligated in frame to a  S. cerevisiae  MNN2(s) [nucleotides 1-108] targeting peptide. The plasmid pPB144 also contains a HYG resistance marker and the  P. pastoris  URA3 gene as targeting sequence for genomic integration. 1 μg plasmids were linearized with SalI, transformed into strain YSH-44 by electroporation, selected on medium containing hygromycin and the resulting strains were screened by analysis of the released glycans from purified K3. Transformants were grown at 30° C. in BMGY to an OD600 of about 100, harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin (Example 3). The N-glycans were released by PNGase digestion (Example 3). The N-glycans were analyzed with a MALDI-TOF MS (Example 3). Transformants exhibiting the transfer of GlcNAc residues onto the Manα1,3 arm of the oligosaccharide structure (e.g. GlcNAc 2 Man 3 GlcNAc 2 ) were designated PBP43 ( FIG. 47 ). Analysis of N-glycans provides a predominant peak at 1543 m/z [y] is consistent with the mass of the glycan GlcNAc 3 Man 3 GlcNAc 2 . 
     Example 26 
     Cloning and Expression of GnTV in  P. pastoris    
     The DNA fragment encoding part of the mouse GnTV protein (MGAT45) lacking the TM domain was PCR amplified from murine cDNA using 
     forward MGV-2 (5′-AAATCAAGTGGATGAAGGACATGTGGC3′) (SEQ ID NO:113) and
 
reverse MGV-3 (5′-AGCGATGCTATAGGCAGTCTTTGCAGAG-3′) (SEQ ID NO:114) primers, cloned into the pCR2.1-TOPO vector (Invitrogen), and sequenced. For cloning into the vector suitable for the fusion with leader library, DNA fragments were PCR amplified with primers that include AscI and Pad restriction sites. AscI forward primers: MGV-ASC1 (5′-TATGGGCGCGCCGATCATAACTCATTGGCGGAAATC-3′) (SEQ ID NO:115), MGV-ASC2 (5′-GAAGGGCGCGCCTTGCCTCCTATGGATGGCTACCCCCAC-3′) (SEQ ID NO:116), MGV-ASC3 (5′-TGGGGCGCGCCGGCAAGCTCGAGTCAAAGGTGGACAAT-3′) (SEQ ID NO:117). Pad reverse primer MGV-PAC (5′-AGTTAATTAATGCTATAGGCAGTCTTTGCAGAG-3′) (SEQ ID NO:118). The nucleic acid (SEQ ID NO:44) and amino acid (SEQ ID NO:43) sequences of mouse GnTV are shown in  FIG. 43 .
 
     Example 27 
       P. pastoris  Strain Expressing GnTV 
       P. pastoris  YSH-44 strain (Example 15) producing complex glycan structures was transformed with the plasmid pPB140 (Table 12) containing a gene fragment encoding the mouse GnTV catalytic domain (Δ45) ligated in frame to a targeting peptide derived from  S. cerevisiae  MNN2(s). Culture conditions were same as in Example 25. The K3 reporter protein from two transformants were analyzed using MALDI-TOF. A peak at 1559 m/z [y] is consistent with the mass of the glycan GlcNAc 3 Man 3 GlcNAc 2  ( FIG. 48 ). A secondary peak at 1355 m/z [u] is consistent with the mass of GlcNAc 2 Man 3 GlcNAc 2 . 
     Example 28 
       P. pastoris  Strain Producing Tetraantennary Structures on Glycoproteins 
       P. pastoris  PBP43 strain (Example 25) producing triantennary glycan structures (e.g., GlcNAc 3 Man 3 GlcNAc 2 ) was transformed with the plasmid pPB140 ( FIG. 40B ) encoding the mouse GnTV (Example 27). The vector pPB140 contains KAN resistance marker and  P. pastoris  HIS3 gene as targeting sequence for genomic integration. 1 μg plasmids were linearized with KpnI, transformed into strain PBP43 by electroporation, selected on medium containing kanomycin and the resulting strains were screened by analysis of the released glycans from purified K3. Culture conditions were same as in Example 25. Analysis of the K3 reporter protein by MALDI-TOF showed a predominant peak at 1747 m/z [z], which is consistent with the mass of the tetraantennary glycan GlcNAc 4 Man 3 GlcNAc 2  ( FIG. 49 ). Hexosaminidase digest (See Example 15) of the resulting glycans showed mass of the peak corresponding to Man 3 GlcNAc 2  (data not shown). 
     In a second experiment,  P. pastoris  YSH-44 was transformed with pPB128 and pPB140 (Table 12). Analysis of the transformants producing the K3 reporter protein by MALDI-TOF showed a predominant peak at 1743 m/z [z], which is consistent with the mass of the tetraantennary glycan GlcNAc 4 Man 3 GlcNAc 2  ( FIG. 50 ). 
     Example 29 
     Cloning and Expression of GnTIX in  P. pastoris    
     The nucleic acid and amino acid sequences of the human GnTIX (AB 109185.1) are shown in  FIG. 45 . The codon optimized DNA fragment encoding part of the human GnTIX lacking the TM domain (Δ43) was synthesized from oligonucleotides using PCR ( FIG. 46 ). The DNA fragment encoding the GnTIX catalytic domain was ligated in frame to a targeting peptide derived from  S. cerevisiae  MNN2(s). The resulting plasmid pPB176 ( FIG. 40C ) was linearized with KpnI and transformed in  P. pastoris  YSH-44 strain (Example 15) producing complex tetraantennary glycan structures. Culture conditions were same as in Example 25. The K3 reporter protein from a transformant was analyzed using MALDI-TOF MS.