Abstract:
Methods and compositions are provided relating to production of recombinant protein in yeast. A modified P LAC4  is described where one or more mutations may be introduced into the Pribnow box-like sequences in the promoter. The modified promoter when placed upstream of a target gene in a vector causes a significant reduction of target gene expression in transformed bacteria but produces efficient expression of the target gene in yeast.

Description:
CROSS REFERENCE 
     This application gains priority from U.S. Provisional Application Ser. No. 60/560,418 filed Apr. 8, 2004, herein incorporated by reference. 
    
    
     BACKGROUND 
     For over a decade, the budding yeast  Kluyveromyces lactis  ( K. lactis ) has been widely used for industrial-scale production of recombinant proteins in the food and dairy industries for reasons that include the following factors: (i) many strains of  K. lactis  grow rapidly and to extremely high cell densities in culture; (ii)  K. lactis  efficiently directs proteins to be secreted into the medium; and (iii)  K. lactis  has GRAS ( G enerally  R egarded  A s  S afe) FDA status which permits its use for food, agricultural and health-related applications. 
     A typical  K. lactis  heterologous protein production strategy involves directing a desired protein to be secreted from the cell into the growth medium. This methodology has a number of advantages over cellular expression methods: (i) the protein is produced significantly pure since  K. lactis  secretes relatively few endogenous proteins; (ii) post-translational protein modifications found only on secreted eukaryotic proteins are obtainable; and (iii) strategies to harvest protein from the medium of continuously growing cells can be devised. 
     A strong yeast promoter suitable for directing high levels of transcription in  K. lactis  is the  K. lactis  LAC4 promoter (P LAC4 ) (Dickson, et al.  Cell  15:123-130 (1978); Dickson, R. C., and M. I. Riley,  Biotechnology  13:19-40 (1989); Dickson, et al.  Mol. Cel. Biol.  1:1048-1056 (1981)). This promoter naturally drives expression of the LAC4 gene which encodes a highly expressed lactase (β-galactosidase). Transcription of LAC4 is elevated in response to the presence of lactose or galactose in growth medium where lactase allows the cell to convert lactose to fermentable sugars. Expression of heterologous proteins from P LAC4  may achieve levels greater than 100 mg L −1  of secreted recombinant protein in yeast fermentations. 
     Unfortunately, in addition to its ability to function as a strong promoter in  K. lactis , P LAC4  constitutively promotes gene expression in  E. coli  cells. This can be particularly problematic when trying to assemble DNA constructs harboring genes that encode a protein toxic to  E. coli  prior to their introduction into yeast cells. One approach to solving this problem has been reported by Gibbs et al. ( FEMS Yeast Research  4: 573-577 (2004)) who utilized yeast introns in the shuttle vector. Unfortunately, this modification abolishes some but not all functional expression of potentially toxic recombinant proteins. 
     SUMMARY 
     In an embodiment of the invention, a method is provided for producing a recombinant protein in yeast cells that includes the steps of: obtaining a vector into which a gene encoding the target protein has been inserted together with a modified P LAC4  wherein the modification results in a significant reduction in gene expression in bacteria exemplified by  E. coli ; transforming yeast cells exemplified by  K. lactis  with the vector; and producing an effective amount of recombinant protein in the yeast cells. In certain embodiments, at least 50%, more particularly at least 70%, more particularly at least 90%, of the transformed yeast cells express recombinant protein. In an embodiment of the invention, the effective amount of recombinant protein produced in yeast is substantially similar to the amount of protein from a recombinant gene under control of an unmodified P LAC4  promoter. 
     The modified P LAC4  in the method may optionally include a mutation in one or more Pribnow box-like sequences, for example in PBI, PBII and PBIII, more particularly in a first region of the promoter corresponding to nucleotides −198 to −212 or in a second region of the promoter corresponding to nucleotides −133 to −146. In certain embodiments, the modified P LAC4  contains one or more mutations in both the first region and also one or more mutations in the second region of the promoter. In a further embodiment of the invention, nucleotides −1 to −283 in the modified P LAC4  are substituted by nucleotides −1 to −283 of the phosphoglycerate kinase promoter from  S. cerevisiae  (PGK1). 
     The vector may be an episomal or an integrative plasmid in the transformed yeast cells. The vector contains a modified P LAC4  promoter and optionally a P LAC4  terminator. Moreover, the vector may include a DNA sequence encoding at least one of a yeast secretion signal peptide such as  K. lactis  α-mating factor (Kl α-MF), a selectable marker such as  Aspergillus nidulans  acetamidase (amdS) selectable marker gene, or a multiple cloning site for insertion of a gene encoding a recombinant protein. 
     The cells transformed with the above-described vector may include a host yeast cell and/or a host bacterial cell. 
     In an embodiment of the invention, a kit that includes a vector as described above and optionally includes competent yeast cells together with instructions for use is provided. 
     An embodiment of the invention provides a modified P LAC4  Pribnow box wherein TTATCATTGT (SEQ ID NO:22) is modified to AGAACAGAGA (SEQ ID NO:23) and/or TATTATTCT is modified to GAGAGCTCT. 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1  shows the  E. coli/K. lactis  integrative expression vector pGBN1. 
       Genes are cloned into the multiple cloning site (MCS) in the same translational reading frame as the  S. cerevisiae  α-mating factor secretion leader sequence (Sc α-MF). Transcription is initiated and terminated by P LAC4 and LAC4 transcription terminator sequence (TT LAC4 ), respectively. The  S. cerevisiae  ADH1 promoter (P ADH1 ) drives expression of a bacterial gene conferring resistance to G418 in yeast.  E. coli  vector sequence has been inserted into a unique SacII site in P LAC4  to allow for propagation in  E. Coli . The vector is linearized by digestion with SacII OR BstXI for integration into the LAC4 promoter locus in the  K. lactis  chromosome. 
         FIG. 2  shows the Pribnow box-like sequences in P LAC4  and construction of P LAC4 variant expression vectors. 
         FIG. 2A  shows Pribnow box-like sequences PBI, and PBII and PBIII (SEQ ID NOS:1 and 2) relative to the major and minor  E. coli  transcription start sites associated with P LAC4 , and are aligned with the Pribnow box consensus sequence TATAAT. Nucleotides that agree with the consensus sequence are boxed. 
         FIG. 2B  shows expression vectors containing P LAC4  variants. The approximate positions of the  E. coli  major and minor transcription start sites are shown in the schematic for pGBN1. The approximate positions of the galactose-responsive elements, upstream activator sequence (UAS) UASI and II, are shown for each construct. Regions of P LAC4  DNA that have been replaced with fragments of the PGK1 promoter are shown in black. Mutated bases in the Pribnow box-like sequences in the P LAC4  DNA of plasmids pGBN1 PBI  and pGBN1 PBII-PBIII  are indicated with a black dot above each base (SEQ ID NOS:3 and 4). All numbered positions are relative to the adenine of the ATG start codon of the Sc α-MF secretion leader that has been designated position +1. 
         FIG. 3  shows P LAC4  variant expression of green fluorescent protein (GFP) in  E. coli  and human serum albumin (HSA) in  K. lactis.    
         FIG. 3A  shows GFP cloned downstream of each of the various P LAC4  variants. Proteins from lysates of  E. coli  carrying each expression construct were separated by SDS-PAGE, and GFP was detected by Western analysis. 
       Lane 1: pGBN1 used as a negative control. Lysate is derived from bacteria containing an empty pGBN1 plasmid; 
       Lane 2: PGBN1/P LAC4  used as a second control containing an unmodified P LAC4 ; 
       Lanes 3-6: lysates used from  E. coli  transformed with pGBN1 in which P LAC4  has been substituted with P PGK1 , P Hybrid , P LAC4-PBI  and P LAC4-PBII-PBIII . 
         FIG. 3B  shows HSA cloned downstream of each P LAC4  for expression in  K. lactis  cells. Secreted proteins in spent culture medium of  K. lactis  strains containing the various integrated HSA expression vectors were resolved by SDS-PAGE (4-20% acrylamide) and Coomassie stained. HSA ran as a single band with an apparent mass of 66 kDa. 
       Lane 1: spent culture medium from a yeast strain containing empty pGBN1 integrated into the chromosome as a negative control; 
       Lanes 2-6: spent media from  K. lactis  transformed with pGBNI LAC4 -HSA, pGBNI PGK1 - HSA, pGBNI Hybrid -HSA, pGBNI LAC4-PBI -HSA and pGBNI LAC4-PBII-PBIII -HSA. 
         FIG. 4  shows pKLAC1, an  E. coil/K. lactis  integrative expression vector. The pKLAC1 vector (GenBank No. AY968582) is organized similarly to pGBN1 with the following modifications: (i) genes are cloned into the multiple cloning site in the same translational reading frame as the native Kl α-MF leader sequence; and (ii) expression in  K. lactis  is initiated by the P LAC4-PBI  variant. The P ADH1  drives expression of an acetamidase-selectable marker (amdS) gene for selection of transformants by growth on acetamide medium. 
         FIG. 5  shows the activity of secreted enterokinase in the spent culture medium of  K. lactis  cells containing integrated pKLAC1-EK L  (the gene encoding the enterokinase catalytic subunit). Seven  K. lactis  strains harboring pKLAC1-EK L  and wild-type GG799 cells were grown in YPGal medium for 48 hours. Cleared spent culture medium was assayed for enterokinase activity by measuring cleavage of a fluorogenic peptide over time. KLEK-S1 and KLEK-S4 are two strains that contain multiple copies of integrated pKLAC1-EK L  as determined by Southern analysis. All other strains contain a single integrated copy of pKLAC1-EK L . 
     
    
    
     DETAILED DESCRIPTION 
     A functional shuttle vector allows for the propagation of cloned genes in bacteria prior to their introduction into yeast cells for expression. However, yeast expression systems that utilize the strong P LAC4  can be adversely affected by the serendipitous expression of protein from genes under control of P LAC4  in bacterial host cells such as  E. coli . This promoter activity can interfere with the cloning efficiency of genes whose translational products are potentially detrimental to bacteria. 
     Two nucleotide sequences in the P LAC4  closely resemble the bacterial Pribnow box transcription element consensus sequence, which is TATAAT. These sequences are located approximately 10 nucleotides upstream from the site where transcription begins and are adjacent and upstream of the major and a minor transcription start sites in  E. coil  (Dickson et al.  Biotechnology  13:19-40 (1989)). In particular, the sequences are located at −204 to −209 for the major transcript, and −144 to −136 for the minor transcript) (see boxed sequences in  FIG. 2A ). 
     The initiation sites of two RNA transcripts associated with  E. coli  expression of  K. lactis  P LAC4  have been previously mapped to −196 bp (initiation of the major  E. coli  transcript) and −127 bp (initiation of the minor  E. coli  transcript) relative to the adenine nucleotide in the ATG start codon of the native LAC4 gene (Dickson et al. 1989). 
     P LAC4  variants with mutated Pribnow box-like sequences can be created by site-directed mutagenesis which substantially retain their ability to function as strong promoters in  K. lactis  to the extent similar to that of unmutated Pribnow box-like sequences. P Lac4  variants that have mutated Pribnow box-like sequences may retain strong promoter activity in other yeast strains from the  Kluyeromyces  species as well as  Saccharomyces  species,  Pichia  species,  Hansenula  species,  Yarrowia  species,  Neurospora  species,  Aspergillus  species,  Penicillium  species,  Candida  species,  Schizosaccharomyces  species,  Cryptococcus  species,  Coprinus  species,  Ustilago  species,  Magnaporth  species and  Trichoderma  species. Based on the knowledge in the art that DNA sequence is determinative for promoter strength, it is expected that some mutants will produce greater amounts of protein than under similar conditions using the wild-type P LAC4 . Mutation is here intended to include any of: a substitution, a deletion or an addition of one or more nucleotides in a DNA sequence. 
     In an embodiment of the invention, the fungal expression host is the yeast  K. lactis  and the bacterial host is  E. coli  and a series of P LAC4  variants have been created by targeted mutagenesis of three DNA sequences (PBI, PBII and PBIII) that resemble the  E. Coli  Pribnow box element of bacterial promoters and that reside immediately upstream of two  E. coli  transcription initiation sites associated with P LAC4 . In the examples, the mutation in P LAC4  is in the region of (a) the −198 to −212 region of the promoter ( FIG. 2B ) for example at positions −201, −203, −204, −207, −209 and −210. These mutations do not substantially interfere with the ability of the promoter to function as a strong promoter in  K. lactis ; (b) the −133 to −146 region of the promoter for example at positions −139, −140, −141, −142 and −144 which do not substantially interfere with strong promoter activity; or (c) the −198 to −212 and −133 to −146 regions. In a further embodiment, a hybrid promoter was created that consists of 283 bp (−1 to −283) of the  S. cerevisiae  (Sc) PGKI promoter replacing the −1 to −283 region of  K. lactis  P LAC4  ( FIG. 2B ). 
     Overexpression of proteins in  K. lactis  and more generally in yeast involves construction of a shuttle vector containing a DNA fragment with sequences suitable for directing high-level transcription of a gene of interest upon introduction into the yeast host. The vector should contain at least one or more of the following: (i) a strong yeast promoter; (ii) DNA encoding a secretion leader sequence (if secretion of the protein into the medium is desired); (iii) the gene encoding the protein to be expressed; (iv) a transcription terminator sequence; and (v) a yeast-selectable marker gene. These sequence components are typically assembled in a plasmid vector in  E. coli  then transferred to yeast cells to achieve protein production. 
     P LAC4  can function as a strong promoter for protein expression in yeast when present on an integrative plasmid or an episomal plasmid such as pKD1-based vectors, 2 micron-containing vectors, and centromeric vectors. The secretion leader sequence (if secretion of the protein into the medium is desired) may include a Sc α-MF pre-pro secretion leader peptide which has been cloned as a HindIII/XhoI fragment. Other prokaryotic or eukaryotic secretion signal peptides (e.g.  K. lactis  α-mating factor pre-pro secretion signal peptide,  K. lactis  killer toxin signal peptide) or synthetic secretion signal peptides can also be used. Alternatively, a secretion leader can be omitted from the vector altogether to achieve cellular expression of the desired protein. 
     An example of a transcription terminator sequence is TT LAC4 . 
     The yeast-selectable marker gene can be for example, G418 or an amdS gene. Expression of acetamidase in transformed yeast cells allows for their growth on medium lacking a simple nitrogen source but containing acetamide. Acetamidase breaks down acetamide to ammonia which can be utilized by cells as a source of nitrogen. A benefit of this selection method is that it enriches transformant populations for cells that have incorporated multiple tandem integrations of a pKLAC1-based expression vector and that produce more recombinant protein than single integrations ( FIG. 5 ). 
     The above-described mutants P LAC4  have been integrated into an  E. coli/K. lactis  integrative shuttle vector, for example, pGBN1 and pKLAC1 shown in  FIGS. 1 and 4 , respectively, which integrates into the  K. lactis  genome after transformation of competent host cells and subsequently directs protein expression. 
     In embodiments of the invention, at least 50%, more specifically at least 70%, preferably at least 90%, of transformants that form on acetamide plates following transformation of  K. lactis  with pKLAC1-based constructs express foreign protein, for example, HSA or the  E. coli  maltose-binding protein (MBP), toxic protease enterokinase, mouse transthyretin, toxic glue proteins from marine organisms and a bacterial cellulase. These examples are not intended to be limiting. The system has utility for any protein-encoding gene placed downstream of the mutated P LAC4 . 
     Levels of protein expression under P LAC4  and mutants thereof were determined for several different proteins. For example, mutation of PBI reduced bacterial expression of a reporter protein (GFP) by ˜87%, whereas mutation of PBII and PBIII had little effect on GFP expression in the bacterial host cell. Deletion of all three sequences completely eliminated GFP expression in the bacterial host cells. For HSA, the Example and  FIG. 3   b  show that about 50 mg L −1  of HSA was secreted by  K. lactis  when expressed from either wild-type or mutant P LAC4 . 
     EXAMPLE 
     Yeast Strains, Transformation and Culturing Conditions 
     The prototrophic  K. lactis  strain GG799 (MAT α [pGK11+]) was routinely grown and maintained on YPD media (1% yeast extract, 2% peptone, 2% glucose) at 30° C. Prior to transformation of GG799 cells, 1 μg of pGBN1- or pKLAC1-based expression vector containing a gene of interest was linearized by SacII digestion. Linearized expression vectors were used for integrative transformation of commercially available competent  K. lactis  GG799 cells (New England Biolabs, Beverly, Mass.) as directed by the supplier. Colonies of cells transformed with pGBN1, pGBN1 PGK1 , pGBN1 Hyb , pGBN1 PBI  or pGBN1 PBII-PBIII  vectors were selected by growth on YPD agar plates containing 200 μg G418 ml −1  (Sigma, St. Louis, Mo.) for 2-3 days at 30° C. Colonies of cells transformed with pKLAC1-based vectors were selected by growth on agar plates containing 1.17% yeast carbon base (New England Biolabs, Beverly, Mass.), 5 mM acetamide (New England Biolabs, Beverly, Mass.) and 30 mM sodium phosphate buffer pH 7 for 4-5 days at 30° C.  K. lactis  strains expressing heterologous genes were cultured in YP media containing 2% galactose (YPGal) at 30° C. for 48-96 hours. 
     Polymerase Chain Reaction 
     Primers used in this study are listed in Table 1. Amplification by PCR was performed using high fidelity Deep Vent™ DNA polymerase (New England Biolabs, Beverly, Mass.). Typical PCR mixtures contained 0.2 mM dNTPs, 0.5 μg of each primer, 1× Thermopol buffer (New England Biolabs, Mass.) and 100 ng template DNA in a total reaction volume of 100 μl. Thermocycling typically consisted of a “hot start” at 95° C. for 5 minutes followed by 30 cycles of successive incubations at 94° C. for 30 sec, 58° C. for 30 sec and 72° C. (1 min per kb of DNA). After thermocycling, a final extension was performed at 72° C. for 10 minutes. 
     Construction of  K. lactis  P LAC4  Variants in pGBN1 
     All promoter variants were derived from wild-type P LAC4  present in the integrative expression vector pGBN1, a  K. lactis/E. coli  shuttle vector that contains 2317 bp of P LAC4  DNA split into 1663 and 654 bp fragments that are separated by pUC19 plasmid DNA ( FIG. 1 ). The split occurs at a unique restriction site recognized by SacII. A 2830 bp of pUC19 vector DNA sequence has been inserted at this unique restriction site. This allows the expression vector to integrate into the promoter region of the native LAC4 locus in the  K. lactis  chromosome after digestion with SacII or BstXI and introduction into yeast cells. Additionally,  K. lactis  DNA that directs integration of the vector into the  K. lactis  chromosome at locations other than LAC4 can be inserted into the vector. Any DNA containing a bacterial origin of replication and a selectable marker gene can be used in place of the pUC19 DNA sequence. The position of the wild-type P LAC4  sequence, or any P LAC4  mutant or hybrid cloned into pGBN1 is immediately upstream of the coding region for the secretion leader sequence. 
     Additionally, pGBN1 contains DNA encoding the Sc α-MF pre-pro domain immediately downstream of P LAC4  to direct secretion of heterologously expressed proteins. Finally, pGBN1 carries a geneticin (G418) resistance gene expressed from the P ADH1  for dominant selection in yeast. To create plasmid pGBN1 PGK1  a PmlI/HindIII fragment containing 488 base pairs of the  S. cerevisiae  PGK1 promoter was cloned into the HpaI/HindIII sites of plasmid pGBN1 to replace 1067 base pairs of native P LAC4  ( FIG. 2B ). Primer P1 and primer P2 were used to amplify 283 base pairs of the  S. cerevisiae  PGK1 promoter using plasmid pGBN1 PGK1  as a template. The 283 bp fragment was cloned into the SnaBI/HindIII sites of plasmid pGBN1 to produce plasmid pGBN1 Hyb . Primer P3 was designed to incorporate mutations into the putative Pribnow box-like sequence (PBI) that lies upstream of the  E. coli  major transcription start site as detailed in  FIG. 2B . Primers P2 and P3 were used to amplify a P LAC4  fragment containing mutations in PBI using plasmid pGBN1 as a template. Amplified DNA from this initial PCR was used as template for a second PCR using primers P2 and P4. The final DNA product was cloned into the SnaBI/HindIII sites of plasmid pGBN1 to produce plasmid pGBN1 PB1 . A PCR knitting method was used to mutate the PBII and PBIII sequences ( FIG. 2B ) that lie upstream of the  E. coli  minor transcription start site using complementary primers P5 and P6. Primers P2 and P5 and primers P4 and P6 were used to amplify 586 bp and 160 bp mutated P LAC4  DNA fragments, respectively. Each amplified DNA product was purified by QiaQuick™ PCR purification spin column chromatography (Qiagen, Valencia, Calif.) and combined as template in a second amplification reaction containing primers P2 and P4. The amplified P LAC4  DNA fragment containing mutagenized PBII and PBIII sites was cloned into the SnaBI/Hind III sites of plasmid pGBN1 to produce plasmid pGBN1 PBII-PBIII . 
     Targeted Mutagenesis of Pribnow Box-Like Sequences in P LAC4    
     A series of four P LAC4  variants were generated to eliminate the  E. coli  promoter activity of P LAC4  by either replacing or introducing point mutations in PBI and PBII/PBIII as shown in  FIG. 2B . 
     (i) Vector pGBN1 pGK1  incorporates 485 bp of the  S. cerevisae  PGK1 promoter (P PGK1 ) in place of 1067 bp of native P LAC4  thereby removing both galactose-responsive upstream activating sequences (UASI and UASII) and all three Pribnow box-like sequences. 
     (ii) Vector pGBN1 Hyb  incorporates 283 bp from the 3′ end of P P PGK1  in place of 283 bp comprising the 3′ end of P LAC4  resulting in deletion of all three Pribnow box-like sequences but leaving both UAS sequences intact. 
     (iii) Vector pGBN1 PB1  contains 6-point mutations that eliminate the Pribnow consensus sequence of PBI between nucleotides −204 and −209 of P LAC4 . 
     (iv) Vector pGBN1 PBII-PBIII  contains 5-point mutations that eliminate the Pribnow consensus sequences of PBII and PBIII between nucleotides −136 and −144 of P LAC4 . 
     Cloning and Expression Analysis of GFP in  E. coli    
     GFP was PCR amplified with primers P7 and P8 using plasmid pGFPuv (Clontech, Palo Alto, Calif.) as a template. Amplified GFP was cloned in-frame with the α-MF pre-pro domain in the BglII/NotI sites of the various PGBN vectors (see previous section). Lysates of bacteria containing various pGBN-GFP constructs were prepared from 50 ml overnight cultures grown at 30° C. in LB medium containing 100 μg/ml ampicillin. Cultures were centrifuged and the cell pellets were frozen on dry ice, thawed at room temperature and resuspended in 10 μl of lysis buffer (20 mM Tris-HCl pH 7.5 containing 50 mM NaCl, 1 mM EDTA). The cells were disrupted with a Sonicator™ (Heat Systems-Ultrasonics, Plainview, N.Y.) for 15 s on setting 7, and cell debris was removed by centrifugation at 15,000×g for 10 minutes. The protein concentration of each lysate was determined by measuring its absorbance at 280 nm. Proteins (100 μg) in each lysate were separated on a Tris-glycine 10-20% SDS-polyacrylamide gel, transferred to nitrocellulose and blocked overnight in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) and 50% non-fat milk (w/v) at 4° C. An anti-GFP monoclonal antibody (Clontech, Palo Alto, Calif.) diluted 1:1000 in PBS-T containing 5% non-fat milk was used to probe the blot followed by incubation with a horseradish peroxidase-coupled anti-mouse secondary antibody (KPL, Gaithersberg, Md.) diluted 1:2000 in PBS-T containing 5% non-fat milk. Protein-antibody complexes were detected using LumiGlo detection reagents (Cell Signaling Technology, Beverly, Mass.). The amount of GFP produced in  E. coli  was measured by densitometry using a molecular imager FX (Bio-Rad, Hercules, Calif.) and Quantity One software. 
     Each P LAC4  variant was tested for its ability to drive  E. coli  expression of a reporter gene encoding GFP that was cloned in-frame with the  S. cerevisiae  α-mating factor pre-pro domain in each of the pGBN vectors. The presence of GFP produced from P LAC4  variants in  E. coli  lysates was analyzed by Western analysis. Removal of the PBI sequence by mutation resulted in an 87% decrease in GFP expression ( FIG. 3A , lane 5), as determined by densitometry, relative to GFP produced by the wild-type P LAC4  ( FIG. 3A , lane 2). However, mutation of both PBII and PBIII sequences ( FIG. 3A , lane 6) did not detectably down-regulate GFP expression. Deletion of all three Pribnow box-like sequences from P LAC4  by replacement with P PGK1  DNA ( FIG. 3A , lanes 3 and 4) lead to a complete loss of detectable GFP expression. These results indicate that the majority of P LAC4  expression in  E. coli  is dependent upon the presence of the PBI sequence. 
     Cloning and Expression of Enterokinase and HSA in  K. lactis    
     Primers P9 and P10 were used to amplify the gene encoding HSA that was subsequently cloned in frame with the α-MF sequence in the XhoI/NotI sites of the various pGBN vectors. Primer P9 was designed to encode the  K. lactis  Kex1 protease cleavage site (KR↓) immediately upstream of the HSA open reading frame to ensure correct processing of the protein in the Golgi.  K. lactis  strains containing integrated pGBN-HSA DNA were grown in 2 ml cultures of YPGal for 48 hours at 30° C. The level of HSA secretion was visually assessed by separation of 15 μl of spent culture medium on 10-20% Tris-Glycine gels followed by Coomassie staining. A DNA fragment encoding the EK L  was PCR amplified with primers P11 and P12 and cloned in-frame with the α-MF pre-pro domain in the XhoI/BglII restriction sites of the various pGBN vectors containing the PLAC4 variants or in the vector pKLAC1 (see below). The DNA sequence of EK L  in the various pGBN-EK L  or pKLAC1-EK L  vectors was confirmed by nucleotide sequencing. Secretion of enterokinase by  K. lactis  strains containing integrated pKLAC1-EK L  constructs was assessed by growing cells in 2 ml YPGal for 48 hours at 30° C. and assaying spent culture medium for enterokinase activity as described below. 
     Enterokinase Activity Assay 
     Spent culture medium was isolated by microcentrifugation of 1 ml of a saturated culture of pKLAC1-EK L  integrated  K. lactis  at 15,800×g for 1 minute to remove cells. Enterokinase activity was measured using the fluorogenic peptide substrate GDDDDK-β-napthylamide (Bachem, King of Prussia, Pa.). Spent culture medium (50 μl) was mixed with 50 μl enterokinase assay buffer (124 mM Tris-HCl pH 8.0 containing 0.88 mM GD4K-β-napthylamide, 17.6% dimethylsulfoxide) and fluorescence intensity (excitation 337 nm, emission 420 nm) was measured over time. A comparison of the amount of enzyme activity associated with measured quantities of purified enterokinase (New England Biolabs, Beverly, Mass.) to the activity present in spent  K. lactis  culture medium was used to estimate the amount of active enterokinase secreted by  K. lactis  strains. To compensate for a mild inhibitory effect that YPGal culture medium has on the enterokinase assay, purified enterokinase was first diluted into spent medium from a culture of untransfected  K. lactis  cells prior to measuring enterokinase activity as described above. 
     P LAC4  Variants Retain Full Promoter Activity in  K. lactis    
     To test if the P LAC4  variants were able to direct expression of a heterologous gene in  K. lactis , the gene encoding HSA was cloned into each of the pGBN vectors. HSA was chosen as a reporter protein due to its high expression and efficient secretion from  K. lactis  when expressed from wild-type P LAC4  (Fleer, et al.  Bio. Technol.  9:968-975 (1991)).  K. lactis  strains containing each of the integrated pGBN1-HSA expression vectors were grown to saturation in YPGal medium and secreted proteins in the spent culture medium were separated by SDS-PAGE and detected by Coomassie staining. HSA migrates as a 66 kDa band that can readily be detected in unconcentrated spent culture medium, and its identity was confirmed by Western blotting with an anti-HSA antibody.  K. lactis  strains containing integrated pGBN1 PB1 -HSA, pGBN1 Hyb -HSA and pGBN1 PBII-PBIII -HSA vectors secreted HSA in amounts comparable to a control strain harboring pGBN1-HSA where HSA is expressed from wild-type P LAC4  ( FIG. 3B , lane 2). These data indicate that mutation or deletion of the PBI, PBII and PBIII sequences of P LAC4  does not significantly alter the promoter&#39;s ability to function in  K. lactis . It is also noteworthy that markedly less HSA was secreted from cells harboring pGBN1 PGK1 -HSA ( FIG. 3B , lane 3) compared to cells expressing HSA from either wild-type P LAC4  ( FIG. 3B , lane 2) or the other P LAC4  variants ( FIG. 3B , lanes 4-6). This is consistent with the notion that HSA expression from P PGK1  is suppressed in galactose-containing medium because both UAS sequences required for galactose-induced expression have been deleted. 
     Effects of P LAC4  Variants on the Cloning Efficiency of Bovine Enterokinase 
     Bovine enterokinase is a commercially important protease that is often used to cleave affinity tags from engineered fusion proteins. Commercial production of enterokinase in  E. coli  is plagued by low yields that are attributable to the protein&#39;s toxicity in bacteria. 
     Expression of enterokinase in  K. lactis  is shown here as a means to circumvent poor expression in bacteria. Numerous attempts to assemble  K. lactis  expression vectors in  E. coli , where DNA encoding the EK L  was placed downstream of wild-type P LAC4 , resulted in widespread isolation of clones containing loss-of-function mutations (e.g. frame shifts or early terminations) within the EK L -coding sequence. P LAC4  variants that exhibited reduced or abolished expression in  E. coli  are shown here to facilitate cloning of the toxic EK L  gene into  K. lactis  expression vectors in  E. coli  prior to their introduction into yeast. The EK L  gene was PCR-amplified using a high-fidelity polymerase and cloned downstream of the various P LAC4  variants in the pGBN1 vectors (see  FIG. 2B ). The entire EK L  gene (708 bp) of numerous isolated clones was sequenced to determine the presence of loss-of-function mutations. When cloned under the control of wild-type P LAC4  in pGBN1, 11 of 12 (92%) clones examined contained loss-of-function mutations. However, no mutations were found in EK L  cloned in vectors pGBN1 PGK1  (9 clones sequenced) or pGBN1 Hyb  (7 clones sequenced), vectors containing P LAC4  variants that completely lack  E. coli  promoter function. Additionally, no mutations were found in EK L  cloned in vector PGBN1 PB1  (9 clones sequenced) where  E. coli  expression is reduced ˜87% due to mutations in PBI. Additionally, 3 of 10 (30%) of EK L  clones in pGBN1 PBII-PBIII  contained loss-of-function mutations. Together, these data show that the function of wild-type P LAC4  in  E. coli  adversely affects the cloning efficiency of a toxic gene, and indicate that P LAC4  variants that either lack or have severely reduced function in  E. coli  are better suited for the assembly of  K. lactis  expression constructs in bacteria. 
     Construction of pKLAC1, an Integrative  K. lactis  Expression Vector 
     A novel  K. lactis  integrative expression vector (pKLAC1) for commercial secretion of proteins from  K. lactis  has been created. This vector is based on the P LAC4-PBI -variant that contains mutations in PBI (see  FIG. 2B , pGBN1 PB1 ) and contains (in 5′ to 3′ order): a PBI-deficient LAC4 promoter, the  K. lactis  α-mating factor secretion leader sequence, a multiple cloning site, the  K. lactis  LAC4 transcription terminator, a selectable marker cassette containing the  Aspergillus nidulans  amdS gene expressed from the P ADH1 , and an  E. coli  origin of replication and ampicillin resistance gene to allow for its propagation in  E. coli.    
     Digestion of this vector with SacII or BstXI generates a linear expression cassette that integrates into the promoter region of the P LAC4  locus of the  K. lactis  chromosome upon its introduction into  K. lactis  cells. Transformed yeast are isolated by nitrogen source selection on yeast carbon base medium containing 5 mM acetamide, which can be converted to a simple nitrogen source only if the expression cassette (containing the amdS gene) has integrated into the chromosome (U.S. Pat. No. 6,051,431). 
     DNA encoding the  K. lactis  α-MF pre-pro domain was PCR-amplified from  K. lactis  genomic DNA using primers 13 and 14 and cloned into the SacI/XhoI sites of pLitmus29 (New England Biolabs, Beverly, Mass.). The cloned  K. lactis  α-MF sequence was subsequently excised by HindIII1 and XhoI digestion and cloned into the HindIII/XhoI sites of plasmid pGBN1 PB1  to produce plasmid pGBN1 PB1 -Kl α-MF. A 1520 bp DNA fragment containing all of the  A. nidulans  amdS gene except the first 128 bp was amplified using primers P15 and P16 and a cloned amdS gene as a template (DSM Biologics B.V., Delft, Netherlands). This fragment was cloned into the BamHI/SmaI sites of plasmid pGBN1 PB1 -Kl α-MF replacing the G418 resistance gene and producing plasmid pGBN1 PB1 -KL α-MF-1520. The remaining 128 bp of the 5′ end of amdS gene was amplified by PCR with primers P16 and P17, digested with BamHI, cloned into the BamHI site of vector pGBN1 PB1 -Kl α-MF-1520 and the proper orientation of the fragment was confirmed by DNA sequencing. The resulting vector is named pKLAC1 (GenBank Accession No. AY968582) and is commercially available from New England Biolabs, Beverly, Mass. 
     Vector pKLAC1 was used to secrete enterokinase from  K. lactis  cells after successfully assembling the expression vector in  E. coli  (pKLAC1-EK L ). Strains harboring integrated pKLAC1-EK L  were cultured in YPGal medium for 2 days. Enterokinase proteolytic activity in the spent culture medium was assayed by measuring the rate of cleavage of a fluorogenic peptide. Measurements of activity performed on culture supernatant from seven pKLAC1-EK L  integrated strains showed that all seven secreted active enterokinase (KLEK) ( FIG. 5 ). However, two of the seven strains (KLEK-S1 and KLEK-S4) secreted greater levels of enterokinase activity than the other five. Southern analysis determined that strains KLEK-S1 and KLEK-S4 contained multiple tandem copies of integrated pKLAC1-EK L . The yield of enterokinase secreted from strain KLEK-S1 grown in shake flasks was estimated to be ˜1.1 mg/L based on a comparison of secreted enzyme activity to the activity of known quantities of purified enterokinase as described above. 
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Oligonucleotides used in this study 
               
             
          
           
               
                 Primer 
                 Sequence* 
               
               
                   
               
             
          
           
               
                 P1 
                 5′-CTGTTACTCTCTCTCTTTCAAACAG-3′ (SEQ ID NO:5) 
                   
               
               
                   
               
               
                 P2 
                 5′-GCATGTATACATCAGTATCTC-3′ (SEQ ID NO:6) 
               
               
                   
               
               
                 P3 
                 5′-GGTATTTAATAGCTCGAATCAATGTGAGAACAGAGAGAAGATGTTCTTCCCTAACTC-3′ 
               
               
                   
                 (SEQ ID NO:7) 
               
               
                   
               
               
                 P4 
                 5′-GTAATGTTTTCATTGCTGTTTTACTTGAGATTTCGATTGAGAAAAAGGTATTTAATAGCTC 
               
               
                   
               
               
                 P5 
                 GAATCAATG-3′ (SEQ ID NO:8) 
               
               
                   
               
               
                 P6 
                 5′-GTTTCTTAGGAGAATGAGAGCTCTTTTGTTATGTTGC-3′ (SEQ ID NO:9) 
               
               
                   
               
               
                 P7 
                 5′-GCAACATAACAAAAGAGCTCTCATTCTCCTAAGAAAC-3′ (SEQ ID NO:10) 
               
               
                   
               
               
                 P8 
                 5′-GGA AGATCT ATGAGTAAAGGAGAAGAACTT-3′ (SEQ ID NO:11) 
               
               
                   
               
               
                 P9 
                 5′-ATAAGAAT GCGGCCGC TTATTTGTAGAGCTCATCCATGCC-3′ (SEQ ID NO:12) 
               
               
                   
               
               
                 P10 
                 5′-CCG CTCGAG AAAAGAGATGCACACAAGAGTGAGGTTGCT-3′ (SEQ ID NO:13) 
               
               
                   
               
               
                 P11 
                 5′-ATAAGAAT GCGGCCGC TTATAAGCCTAAGGCAGC-3′ (SEQ ID NO:14) 
               
               
                   
               
               
                 P12 
                 5′-CCG CTCGAG AAAAGAATTGTTGGTGGTTCTGATTCTAGA-3′ (SEQ ID NO:15) 
               
               
                   
               
               
                 P13 
                 5′-GGA AGATCT CTAATGTAGAAAACTTTGTATCC-3′ (SEQ ID NO:16) 
               
               
                   
               
               
                 P14 
                 5′-TCC GAGCTCAAGCTT GAAAAAAATGAAATTCTCTACTATATTAGCC-3′ (SEQ ID NO:17) 
               
               
                   
               
               
                 P15 
                 5′-CCG CTCGAG ATCATCCTTGTCAGCGAAAGC-3′ (SEQ ID NO:18) 
               
               
                   
                 5′-CGG GGATCC TTTCAGAGGCCGAACTGAAGATCACAGAGGCTTCCGCTGCGGATCTTGTG 
               
               
                   
               
               
                 P16 
                 TCCAAGCTGGCGGCCGGA-3′ (SEQ ID NO:19) 
               
               
                   
               
               
                 P17 
                 5′-TCC CCCGGG CTATGGAGTCACCACATTTCCCAGCAA-3′ (SEQ ID NO:20) 
               
               
                   
                 5′-CGC GGATCC GCCACCATGCCTCAATCCTGGGAAGAA-3′ (SEQ ID NO:21) 
               
               
                   
               
               
                 *Engineered restriction sites are underlined.