Abstract:
The invention relates to DNA molecules, recombinant vectors and cell cultures for use in methods for expression of bile salt-stimulated lipase (BSSL) in the methylotrophic yeast  Pichia pastoris.

Description:
TECHNICAL FIELD  
         [0001]    The invention relates to DNA molecules, recombinant vectors and cell cultures for use in methods for expression of bile salt-stimulated lipase (BSSL) in the methylotrophic yeast  Pichia pastoris.    
         BACKGROUND ART  
         [0002]    Bile salt-stimulated lipase (BSSL; EC 3.1.1.1) (for a review see Wang &amp; Hartsuck, 1993) accounts for the majority of the lipolytic activity of the human milk. A characteristic feature of this lipase is that it requires primary bile salts for activity against emulsified long chain triacylglycerols. BSSL has so far been found only in milk from man, gorilla, cat and dog (Hernell et al., 1989).  
           [0003]    BSSL has been attributed a critical role for the digestion of milk lipids in the intestine of the breastfed infant (Fredrikzon et al., 1978). BSSL is synthesized in humans in the lactating mammary gland and secretes with milk (Bläckberg et al., 1987). It accounts for approximately 1% of the total milk protein (Bläckberg &amp; Hernell, 1981).  
           [0004]    It has been suggested that BSSL is the major rate limiting factor in fat absorption and subsequent growth by, in particular premature, infants who are deficient in their own production of BSSL, and that supplementation of formulas with the purified enzyme significantly improves digestion and growth of these infants (U.S. Pat. No. 4,944,944; Oklahoma Medical Research Foundation). This is clinically important in the preparation of infant formulas which contain relative high percentage of triglycerides and which are based on plant or non human milk protein sources, since infants fed with these formulas are unable to digest the fat in the absence of added BSSL.  
           [0005]    The cDNA structures for both milk BSSL and pancreas carboxylic ester hydrolase (CEH) have been characterized (Baba et al., 1991; Hui and Kissel, 1991; Nilsson et al., 1991; Reue et al., 1991) and the conclusion has been drawn that the milk enzyme and the pancreas enzyme are products of the same gene, the CEL gene. The cDNA sequence (SEQ ID NO: 1) of the CEL gene is disclosed in U.S. Pat. No. 5,200,183 (Oklahoma Medical Research Foundation); WO 91/18293 (Aktiebolaget Astra); Nilsson et al., (1990); and Baba et al., (1991). The deduced amino acid sequence of the BSSL protein, including a signal sequence of 23 amino acids, is shown as SEQ ID NO: 2 in the Sequence Listing, while the sequence of the native protein of 722 amino acids is shown as SEQ ID NO: 3.  
           [0006]    The C-terminal region of the protein contains 16 repeats of 11 amino acid residues each, followed by an 11 amino acid conserved stretch. The native protein is highly glycosylated and a large range of observed molecular weights have been reported. This can probably be explained by varying extent of glycosylation (Abouakil et al., 1988). The N-tenninal half of the protein is homologous to acetyl choline esterase and some other esterases (Nilsson et al., 1990).  
           [0007]    Recombinant BSSL can be produced by expression in a suitable host such as  E. coli, Saccharomyces cerevisiae , or mammalian cell lines. For the scaling-up of a BSSL expression system to make the production cost commercially viable, utilization of heterologous expression systems could be envisaged. As mentioned above, human BSSL has 16 repeats of 11 amino acids at the C-terminal end. To determine the biological significance of this repeat region, various mutants of human BSSL have been constructed which lack part or whole of the repeat regions (Hansson et al., 1993). The variant BSSL-C (SEQ ID NO: 4), for example, has deletions from amino acid residues 536 to 568 and from amino acid residues 591 to 711. Expression studies, using mammalian cell line C127 host and bovine papilloma virus expression vector, showed that the various variants can be expressed in active forms (Hansson et al., 1993). From the expression studies it was also conduded that the proline rich repeats in human BSSL are not essential for catalytic activity or bile salt activation of BSSL. However, production of BSSL or its mutants in a mammalian expression system could be too expensive for routine therapeutic use.  
           [0008]    A eukaryotic system such as yeast may provide significant advantages, compared to the use of prokaryotic systems, for the production of certain polypeptides encoded by recombinant DNA. For example, yeast can generally be grown to higher cell densities than bacteria and may prove capable of glycosylating expressed polypeptides, where such glycosylation is important for the biological activity. However, use of the yeast  Saccharomyces cerevisiae  as a host organism often leads to poor expression levels and poor secretion of the recombinant protein (Cregg et al., 1987). The maximum levels of heterologous proteins in  S. cerevisae  are in the region of 5% of total cell protein (Kingsman et al., 1985). A further drawback of using  Sacharomyces cerevisiae  as a host is that the recombinant proteins tend to be overglycosylated which could affect activity of glycosylated mammalian proteins.  
           [0009]    [0009] Pichia pastoris  is a methylotrophic yeast which can grow on methanol as a sole carbon and energy source as it contains a highly regulated methanol utilization pathway (Ellis et al., 1985).  P. pastoris  is also amenable to efficient high cell density fermentation technology. Therefore recombinant DNA technology and efficient methods of yeast transformation have made it possible to develop  P. pastoris  as a host for expression of heterologous protein in large quantity, with a methanol oxidase promoter based expression system (Cregg et al., 1987).  
           [0010]    Use of  Pichia pastoris  is known in the art as a host for the expression of e.g. the following heterologous proteins: human tumor necrosis factor (EP-A-0263311); Bordetella pertactin antigens (WO 91/15571); hepatitis B surface antigen (Cregg et al., 1987); human lysozyme protein (WO 92/04441); aprotinin (WO 92/01048). However, successful expression of a heterologous protein in active, soluble and secreted form depends on a variety of factors, e.g. correct choice of signal peptide, proper construction of the fusion junction between the signal peptide and the mature protein, growth conditions, etc.  
         PURPOSE OF THE INVENTION  
         [0011]    The purpose of the invention is to overcome the above mentioned drawbacks with the previous systems and to provide a method for the production of human BSSL with is cost-effective and has a yield comparable with, or superior to, production in other organisms. This purpose has been achieved by providing methods for expression of BSSL in  Pichia pastoris  cells.  
           [0012]    By the invention it has thus been shown that human BSSL and the variant BSSL can be expressed in active form secreted from  P. pastoris . The native signal peptide, as well as the heterologous signal peptide derived from  S. cerevisiae  invertase protein, have been used to transiocate the mature protein into the culture medium as an active, properly processed form.  
         DESCRIPTION OF THE INVENTION  
         [0013]    In a first aspect, the invention provides a DNA molecule comprising:  
           [0014]    (a) a region coding for a polypeptide which is human BSSL or a biologically active variant thereof;  
           [0015]    (b) joined to the 5′-end of said polypeptide coding region, a region coding for a signal peptide capable of directing secretion of said polypeptide from  Pichia pastoris  cells transformed with said DNA molecule; and  
           [0016]    (c) operably-linked to said coding regions defined in (a) and (b), the methanol oxidase promoter of  Pichia pastoris  or a functionally equivalent promoter.  
           [0017]    The term “biologically active variant” of BSSL is to be understood as a polypeptide having BSSL activity and comprising part of the amino acid sequence shown as SEQ ID NO: 3 in the Sequence Listing. The term “polypeptide having BSSL activity” is in this context to be understood as a polypeptide comprising the following properties: (a) being suitable for oral administration; (b) being activated by specific bile-salts; and (c) acting as a non-specific lipase in the contents of the small intestines, i.e. being able to hydrolyze lipids relatively independent of their chemical structure and physical state (emulsified, micellar, soluble).  
           [0018]    The said BSSL variant can e.g. be a variant which comprises less than 16 repeat units, whereby a “repeat unit” will be understood as a repeated unit of 11 amino acids, encoded by a nudeotide sequence indicated as a “repeat unit” under the heading “(ix) FEATURE” in “INFORMATION FOR SEQ ID NO: 1” in the Sequence Listing. In particular, the BSSL variant can be the variant BSSL-C, wherein amino acids 536 to 568 and 591 to 711 have been deleted (SEQ ID NO: 4 in the Sequence Listing). Consequently, the DNA molecule according to the invention is preferably a DNA molecule which encodes BSSL (SEQ ID NO: 3) or BSSL-C (SEQ ID NO: 4).  
           [0019]    However, the DNA molecules according to the invention are not to be limited strictly to DNA molecules which encode polypeptides with amino acid sequences identical to SEQ ID NO: 3 or 4 in the Sequence Listing. Rather the invention encompasses DNA molecules which code for polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of BSSL. Included in the invention are consequently DNA molecules coding for BSSL variants as stated above and also DNA molecules coding for polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence shown as SEQ ID NO: 3 or 4 in the Sequence Listing.  
           [0020]    The signal peptide referred to above can be a peptide which is identical to, or substantially similar to, the peptide with the amino acid sequence shown as amino acids −20 to −1 of SEQ ID NO: 2 in the Sequence Listing. Alternatively, it can be a peptide which comprises a  Saccharomyces cerevisiae  invertase signal peptide.  
           [0021]    In a further aspect, the invention provides a vector comprising a DNA molecule as defined above. Preferably, such a vector is a replicable expression vector which carries and is capable of mediating expression, in a cell of the genus Pichia, of a DNA sequence coding for human BSSL or a biologically active variant thereof. Such a vector can e.g. be the plasmid vector pARC 5771 (NCIMB 40721), pARC 5799 (NCIMB 40723) or pARC 5797 (NCIMB 40722).  
           [0022]    In another aspect, the invention provides a host cell culture comprising cells of the genus Pichia transformed with a DNA molecule or a vector as defined above. Preferably, the host cells are  Pichia pastoris  cells of a strain such as PPF-1 or GS115. The said cell culture can e.g. be the culture PPF-1[pARC 5771] (NCIMB 40721), GS115[pARC 5799] (NCIMB 40723) or GS115[pARC 5797] (NCIMB 40722).  
           [0023]    In yet another aspect, the invention provides a process the production of a polypeptide which is human BSSL, or a biologically active variant thereof, which comprises culturing host cells according to the invention under conditions whereby said polypeptide is secreted into the culture medium, and recovering said polypeptide from the culture medium. 
       
    
    
     EXAMPLES OF THE INVENTION  
     Example 1  
     Expression of BSSL in  Pichia pastoris  PPF-1  
       [0024]    1.1. Construction of pARC 0770  
         [0025]    The cDNA sequence (SEQ ID NO: 1) coding for the BSSL protein, including the native signal peptide (below referred to as NSP) was cloned in pTZ19R (Pharmacia) as an EcoRI-SacI fragment. The cloning of NSP-BSSL cDNA into  S. cerevisiae  expression vector pSCW 231 (obtained from professor L. Prakash, University of Rochester, N.Y., USA), which is a low copy number yeast expression vector wherein expression is under control of the constitutive ADH1 promoter, was achieved in two steps. Initially the NSP-BSSL cDNA was cloned into pYES 2.0 (Invitrogen, USA) as an EcoRI-SphI fragment from pTZ19R-SP-BSSL. The excess 89 base pairs between the EcoRI and NcoI at the beginning of the signal peptide coding sequence were removed by creating an EcoRI/NcoI (89) fusion and regenerating an EcoRI site. The resulting clone pARC 0770 contained an ATG codon, originally encoded within the NcoI site which was immediately followed by the regenerated EcoRI site in frame with the remaining NSP-BSSL sequence.  
         [0026]    1.2. Construction of pARC 5771 Plasmid  
         [0027]    To construct a suitable expression vector for the expression of BSSL, the cDNA fragment encoding the BSSL protein along with its native signal peptide was cloned with  P. pastoris  expression vector pDM 148. The vector pDM 148 (received from Dr. S. Subramani, UCSD) was constructed as follows: the upstream untranslated region (5′-UTR) and the down stream untranslated region (3′-UTR) of methanol oxidase (MOX1) gene were isolated by PCR and placed in tandem in the multiple cloning sequence (MCS) of  E. coli  vector pSK +  (available from Stratagene, USA).  
         [0028]    For proper selection of the putative  P. pastoris  transformants, a DNA sequence coding for  S. cerevisiae  ARG4 gene along with its own promoter sequence was inserted between the 5′- and the 3′-UTR in pSK−. The resulting construct pDM148 has following features: in the MCS region of pSK− the 5′-UTR of MOX,  S. cerevisiae  ARG4 genomic sequence and the 3′-UTR of MOX were cloned. Between the 5′-UTR of MOX and the ARG4 genomic sequence a series of unique restriction sites (SalI, ClaI, EcoRI, PstI, SmaI and BamHI) were situated where any heterologous protein coding sequence can be cloned for expression under the control of the MOX promoter in  P. pastoris . To facilitate integration of this expression cassette into the MOX1 locus in  P. pastoris  chromosome, the expression cassette can be cleaved from the rest of the pSK −  vector by digestion with NotI restriction enzyme.  
         [0029]    The 5′-UTR of MOX1 of  P. pastoris  cloned in pDM 148 was about 500 bp in length while the 3′-UTR of MOX1 from  P. pastoris  cloned into pDM 148 was about 1000 bp long. To insert the NSP-BSSL cDNA sequence, between the 5′-UTR of MOX1 and the  S. cerevisiae ARG 4 coding sequence in pDM 148, the cDNA insert (SP-BSSL) was isolated from pARC 0770 by digestion with EcoRI and BamHI (approximately 2.2 kb DNA fragment) and cloned between the EcoRI and BamHI sites in pDM 148.  
         [0030]    The resulting construct pARC 5771 (NCIMB 40721) contained the  P. pastoris  MOX1 5′-UTR followed by the NSP-BSSL coding sequence followed by  S. cerevisiae  ARG4 gene sequence and 3′-UTR of MOX1 gene of  P. pastoris  while the entire DNA segment from 5′-UTR of MOX1 to the 3′-UTR of MOX1 was cloned at the MCS of pSK−.  
         [0031]    1.3. Transformation of BSSL in  P. pastoris  Host PPF-1  
         [0032]    For expression of BSSL in  P. pastoris  host PPF-1 (his4, arg4; received from Phillips Petroleum Co.), the plasmid pARC 5771 was digested with NotI and the entire digested mix (10 μg of total DNA) was used to transform PPF-1. The transformation protocol followed was essentially the yeast spheroplast method described by Cregg et al. (1987). Transformants were regenerated on minimal medium lacking arginine so that Arg+ colonies could be selected. The regeneration top agar containing the transformants was lifted and homogenized in water and yeast cells plated to about 250 colonies per plate on minimal glucose plates lacking arginine. Mutant colonies are then identified by replica plating onto minimal methanol plates. Approximately 15% of all transformants turned out to be Mut s  (methanol slow growing) phenotype.  
         [0033]    1.4. Screening for Transformants Expressing BSSL  
         [0034]    In order to screen large number of transformants rapidly for the expression of lipase a lipase plate assay method was developed. The procedure for preparing these plates was as follows: to a solution of 2% agarose (final), 10×Na-cholate solution in water was added to a final concentration of 1%. The lipid substrate trybutine was added in the mixture to a final concentration of 1% (v/v). To support growth of the transformants the mixture was further supplemented with 0.25% yeast nitrogen base (final) and 0.5% methanol (final). The ingredients were mixed properly and poured into plates up to 3-5 mm thickness. Once the mixture became solid, the transformants were streaked onto the plates and the plates were further incubated at +37° C. for 12 h. The lipase producing clones showed a clear halo around the clone. In a typical experiment 7 out of a total of 93 transformants were identified as BSSL producing transformants. Two clones (Nos. 39 and 86) producing the largest halos around the streaked colony were picked out for further characterization.  
         [0035]    1.5. Expression of BSSL from PPF-1[pARC 5771] 
         [0036]    The two transformants Nos. 39 and 86 described in Section 1.4 were picked out and grown in BMGY liquid media (1% yeast extract, 2% bactopeptone, 1.34% yeast nitrogen base without amino acid, 100 mM KPO 4  buffer, pH 6.0, 400 μg/l biotin, and 2% glycerol) for 24 h at 30° C. until the cultures reached A 600  close to 40. The cultures were pelleted down and resuspended in BMMY (2% glycerol replaced by 0.5% methanol in BMGY) media at A 600 =300. The induced cultures were incubated at 30° C. with shaking for 120 h. The culture supernatants were withdrawn at different time points for the analysis of the expression of BSSL by enzyme activity assay, SDS-PAGE analysis and western blotting.  
         [0037]    1.6. Detection of BSSL Enzyme Activity in the Culture Supernatants of Clone Nos. 39 and 86  
         [0038]    To determine the enzyme activity in the cell free culture supernatant of the induced cultures Nos. 39 and 86 as described in Section 1.5, the cultures were spun down and 2 μl of the cell free supernatant was assayed for BSSL enzyme activity according to the method described by Hernell and Olivecrona (1974). As shown in Table 1, both the cultures were found to contain BSSL enzyme activity with the maximum activity at 96 h following induction.  
         [0039]    1.7. Western Blot Analysis of Culture Supernatants of PPF-1:pARC 5771 Transformants (Nos. 39 and 86)  
         [0040]    To determine the presence of recombinant BSSL in the culture supernatants Nos. 39 and 86 of PPF-1[pARC 5771] transformants, the cultures were grown and induced as described in Section 1.5. The cultures were withdrawn at different time points following induction and subjected to Western blot analysis using anti BSSL polyclonal antibody. The results indicated the presence of BSSL in the culture supernatant as a 116 kDa band.  
       Example 2  
     Expression of BSSL in  Pichia pastoris  GS115  
       [0041]    2.1. Construction of pARC 5799  
         [0042]    Since the 5′-MOX UTR and 3′-MOX UTR were not properly defined and since the pDM 148 vector lacks any other suitable marker (e.g. a G418 resistance gene) to monitor the number of copies of the BSSL integrated in the Pichia chromosome, the cDNA insert of native BSSL along with its signal peptide was cloned into another  P. pastoris  expression vector, pHIL D4. The integrative plasmid pHIL D4 was obtained from Phillips Petroleum Company. The plasmid contained 5′-MOX1, approximately 1000 bp segment of the alcohol oxidase promoter and a unique EcoRI doning site. It also contained approximately 250 bp of 3′-MOX1 region containing alcohol oxidase terminating sequence, following the EcoRI site. The “termination” region was followed by  P. pastoris  histidinol dehydrogenase gene HIS4 contained on a 2.8 kb fragment to complement the defective HIS4 gene in the host GS115 (see below). A 650 bp region containing 3′-MOX1 DNA was fused at the 3′-end of HIS4 gene, which together with the 5′-MOX1 region was necessary for site-directed integration. A bacterial kanamycin resistance gene from pUC4K (PL-Biochemicals) was inserted at the unique NaeI site between HIS4 and 3′-MOX1 region at 3′ of the HIS4 gene.  
         [0043]    To clone the NSP-BSSL coding cDNA fragment at the unique EcoRI site of pHIL D4, a double stranded oligo linker having a BamHI-EcoRI cleaved position was ligated to the BamHI digested plasmid pARC 5771 and the entire NSP-BSSL coding sequence was pulled out as a 2.2 kb EcoRI fragment. This fragment was cloned at the EcoRI site of pHIL D-4 and the correctly oriented plasmid was designated as pARC 5799 (NCIMB 40723).  
         [0044]    2.2. Transformation of pARC 5799  
         [0045]    To facilitate integration of the NSP-BSSL coding sequence at the genomic locus of MOX1 in  P. pastoris  the plasmid pARC 5799 was digested with BglII and used for transformation of  P. pastoris  strain GS115(his4) (Phillips Petroleum Company) according to a protocol described in Section 1.5. In this case, however, the selection was for His prototrophy. The transformants were picked up following serial dilution plating of the regenerated top agar and tested directly for lipase plate assay as described in Section 1.4. Two transformant clones (Nos. 9 and 21) were picked up on the basis of the halo size on the lipase assay plate and checked further for the expression of BSSL. The clones were found to be Mut + .  
         [0046]    2.3. Determination of BSSL Enzyme Activity in the Culture Supernatants of GS115[pARC 5799] Transformants Nos. 9 and 21.  
         [0047]    The two transformed clones Nos. 9 and 21 of GS115[pARC 5799] were grown essentially following the protocol described in Section 1.5. The culture supernatants at different time points following induction were assayed for BSSL enzyme activity as described in Section 1.6. As shown in Table 1, both the culture supernatants were found to contain BSSL enzyme activity and the enzyme activity was highest after 72 h of induction. Both clones showed a superior expression of BSSL compared to the clones of PPF-1[pARC 5771].  
         [0048]    [0048] 2 . 4 . SDS-PAGE and Western Blot Analysis of Culture Supernatants of GS115[pARC 5799] Transformants Nos. 9 and 21  
         [0049]    The culture supernatants collected at different time points, as described in Section 2.3 were subjected to SDS-PAGE and western blot analysis. From the SDS-PAGE profile it was estimated that about 60-75% of the total protein present in the culture supernatants of the induced cultures was BSSL. The molecular weight of the protein was about 116 kDa. The western blot data also confirmed that the major protein present in the culture supernatant was BSSL. The protein apparently had the same molecular weight as the native BSSL.  
       Example 3  
     Scaling-Up of BSSL Expression  
       [0050]    3.1. Scaling-up of Expression of BSSL from the Transformed Clone GS115[pARC 5799] (No. 21)  
         [0051]    A 23 l capacity B. Braun fermenter was used. Five liters of medium containing, 1% YE, 2% Peptone, 1.34 YNB and 4% w/v glycerol was autoclaved at 121° C. for 30 mm and biotin (400 μg/L final concentration) was added during inoculation after filter sterilization. For inoculum, glycerol stock of GS115[pARC 5799] (No. 21) inoculated into a synthetic medium containing YNB (67%) plus 2% glycerol (150 ml) and grown at +30° C. for 36 h was used. Fermentation conditions were as follows: the temperature was +30° C.; pH 5.0 was maintained using 3.5 N NH 4 OH and 2 N HCl; dissolved oxygen from 20 to 40% of air saturation; polypropylene glycol 2000 was used as antifoam.  
         [0052]    Growth was monitored at regular intervals by taking OD at 600 nm. A 600  reached a maximum of 50-60 in 24 h. At this point, the batch growth phase was over as indicated by the increased dissolved oxygen levels.  
         [0053]    Growth phase was immediately followed by the induction phase. During this phase, methanol containing 12 ml/L PTM1 salts was fed. Methanol feed rate was 6 μl/h during first 10-12 h after which it was increased gradually in 6 ml/h increments every 7-8 h to a maximum of 36 ml/h. Ammonia used for pH control acted as a nitrogen source. Methanol accumulation was checked every 6-8 h by using dissolved oxygen spiking and it was found to be limiting during the entire phase of induction. OD at 600 nm increased from 50-60 to 150-170 during 86 h of methanol feed. Yeast extract and peptone were added every 24 h to make final conc. of 0.25% and 0.5% respectively.  
         [0054]    Samples were withdrawn at 24 h interval and checked for BSSL enzyme activity in the cell free broth. The broth was also subjected to SDS-PAGE and western blotting analysis.  
         [0055]    3.2. Protein Analysis of the Secreted BSSL from the Fermenter Grown Culture GS115[pARC 5799] (No. 21)  
         [0056]    BSSL enzyme activity in cell free broth increased from 40-70 mg/l (equivalent of native protein) in 24 h to a maximum 200-227.0 mg/l (equivalent of native protein) at the end of 86-90 h. SDS-PAGE analysis of the cell free broth shows a prominent coomassie blue stained band of mol.wt. of 116 kDa. The identity of the band was confirmed by Western blot performed as described in Section 1.7 for native BSSL.  
         [0057]    3.3. Purification of Recombinant BSSL Secreted into the Culture Supernatant of GS115[pARC 5799] (No. 21) Clones  
         [0058]    The  P. pastoris  clone GS115[pARC 5799] was grown and induced in the fermenter as described in Section 3.1. For purification of recombinant BSSL, 250 ml of culture medium (induced for 90 h) was spun at 12,000× g for 30 minutes to remove all particulate matter. The cell free culture supernatant was ultra filtered in an Amicon set up using a 10 kDa cut off membrane. Salts and low molecular weight proteins and peptides of the culture supernatant were removed by repeated dilution during filtration. The buffer used for such dilution was 5 mM Barbitol pH 7.4. Following concentration of the culture supernatant, the retentate was reconstituted to 250 ml using 5 mM Barbitol, pH 7.4 and 50 mM NaCl and loaded onto a Heparin-Sepharose column (15 ml bed volume) which was pre-equilibrated with the same buffer. The sample loading was done at a flow rate of 10 ml/hr. Following loading the column was washed with 5 mM Barbitol, pH 7.4 and 0.1 M NaCl (200 μl washing buffer) till the absorbance at 250 nm reached below detection level. The BSSL was eluted with 200 ml of Barbitol buffer (5 mM, pH 7.4) and a linear gradient of NaCl ranging from 0.1 M to 0.7 M. Fractions (2.5 ml) were collected and checked for the eluted protein by monitoring the absorbance at 260 nm. Fractions containing protein were assayed for BSSL enzyme activity. Appropriate fractions were analyzed on 8.0% SDS-PAGE to check thee purification profile.  
         [0059]    3.4. Characterization of Purified Recombinant BSSL Secreted in the Culture Supernatant of GS115[pARC 5799] 
         [0060]    SDS-PAGE and Western blot analysis of the fractions (described in Section 3.3) showing maximal BSSL enzyme activity demonstrated that the recombinant protein was approximately 90% pure. The molecular weight of the purified protein was about 116 kDa as determined by SDS-PAGE and western blot analysis. When the samples were overloaded for SDS-PAGE analysis a low molecular weight protein band could be detected by Coomassie Brilliant Blue staining which was not picked up on Western blot. The purified protein was subjected to N-terminal analysis in an automated protein sequencer. The results showed that the protein was properly processed from the native signal peptide and the recombinant protein has the N-terminal sequence A K L G A V Y. The specific activity of the purified recombinant protein was found to be similar to that of the native protein.  
       Example 4  
     Expression of BSSL-C in  Pichia pastoris  GS115  
       [0061]    4.1. Construction of pARC 5797  
         [0062]    The cDNA coding sequence for the BSSL variant BSSL-C was fused at its 5′-end with the signal peptide coding sequence of  S. cerevisiae  SUC2 gene product (invertase), maintaining the integrity of the open reading frame initiated at the first ATG codon of invertase signal peptide. This fusion gene construct was initially cloned into the  S. cerevisiae  expression vector pSCW 231 (pSCW 231 is a low copy number yeast expression vector and the expression is under the control of the constitutive ADH1 promoter) between EcoRI and BamHI site to generate the expression vector pARC 0788.  
         [0063]    The cDNA of the fusion gene was further subdoned into  P. pastoris  expression vector pDM 148 (described in Section 1.2) by releasing the appropriate 1.8 kb fragment by EcoRI and BamHI digestion of pARC 0788 and subcloning the fragment into pDM 148 digested with EcoRI and BamHI. The resulting construct pARC 5790 was digested with BamHI. and a double stranded oligonudeotide linker of the physical structure BamHI-EcoRI-BamHI was ligated to generate the construct pARC 5796 essentially to isolate the cDNA fragment of the fusion gene, following the strategy as described in Section 2.1.  
         [0064]    Finally the 1.8 kb fragment containing the invertase signal peptide/BSSL-C fusion gene was released from pARC 5796 by EcoRI digestion and cloned into pHIL D4 at the EcoRI site. By appropriate restriction analysis of the expression vector containing the insert in the proper orientation was identified and was designated as pARC 5797 (NCIMB 40722).  
         [0065]    4.2. Expression of Recombinant BSSL-C from  P. pastoris    
         [0066]    To express recombinant BSSL-C from  P. pastoris , the  P. pastoris  host GS115 was transformed with pARC 5797 by the method as described in Sections 1.3 and 2.2. Transformants were checked for lipase production by the method described in Sections 1.4 and 2.2. A single transformant (No. 3) was picked on the basis of high lipase producing ability by the lipase plate assay detection method and was further analyzed for production of BSSL enzyme activity in the culture supernatant by essentially following the method as described in Sections 1.6 and 2.3. As shown in Table 1, the culture supernatant of GS115[pARC 5797] (No. 3) contained BSSL enzyme activity and the amount increased progressively till 72 h following induction.  
         [0067]    4.3. SDS-PAGE and Western Blot Analysis of Culture Supernatant of GS115[pARC 5797] Transformant (No. 3)  
         [0068]    The culture supernatant collected at various time points as described in Section 4.2 were subjected to SDS-PAGE and western blot analysis as described in Sections 1.7 and 2.4. From the SDS-PAGE profile it was estimated that about 75-80% of the total extracellular protein was BSSLC. The molecular weight of the protein as estimated from SDS-PAGE analysis was approximately 66 kDa. On western blot analysis only two bands (doublet) around 66 kDa were found to be immunoreactive and thus confirming the expression of recombinant BSSL-C.  
       Example for Comparison  
     Expression of BSSL in  S. cerevisiae    
       [0069]    Attempts to express BSSL in  Saccharomyces cerevisiae  were made. BSSL was poorly secreted in  S. cerevisiae  and the native signal peptide did not work efficiently. In addition, the native signal peptide did not get cleaved from the mature protein in  S. cerevisiae.    
       REFERENCES  
       [0070]    Abouakil, N., Rogalska, E., Bonicel, J. and Lombardo, D. (1988) Biochim. Biophys. Acta. 961, 299-308.  
         [0071]    Baba, T., Downs, D., Jackson, K. W., Tang, J. and Wang, C -S (1991) Biochemistry 30, 500-510.  
         [0072]    Bläckberg, L. and Hernell, O. (1981) Eur. J. Biochem. 116, 221-225.  
         [0073]    Bläckberg, L., Ängquist, K. A. and Hernell, O. (1987) FEBS Lett. 217, 37-41.  
         [0074]    Cregg, J. M. et al. (1987) Bio/Technology 5, 479-485.  
         [0075]    Ellis, S. B. et al. (1985) Mol. Cell. Biol. 5, 1111-1121.  
         [0076]    Fredrikzon, B., Hernell, O., Bläckberg, L. and Olivecrona, T. (1978) Pediatric Res. 12, 1048-1052.  
         [0077]    Hansson, L., Bläckberg, L., Edlund, M., Lundberg, L., Strömqvist, M. and Hernell, O. (1993) J. Biol. Chem. 268, 26692-26698.  
         [0078]    Hernell, O . and Olivecrona, T. (1974) Biochim. Biophys. Acta 369, 234-244.  
         [0079]    Hernell, O., Bläckberg, L and Olivecrona, T. (1989) in: Textbook of gastroenterology and nutrition in infancy (Lebenthal, E., ed.) 347-354, Raven Press, NY.  
         [0080]    Hernell, O. and Bläckberg, L. (1982) Pediatric Res. 16, 882-885.  
         [0081]    Hui, D. Y. and Kissel, J. A. (1990) FEBS Letters 276, 131-134.  
         [0082]    Kingsman, et. al. (1985) Biotechnology and Genetic Engineering Reviews 3, 377-416.  
         [0083]    Nilsson, J., Bläckberg, L., Carlsson, P., Enerbäck, S., Hernell, O. and Bjursell, G. (1990) Eur. J. Biochem. 192, 543-550.  
         [0084]    Reue, K., Zambaux, J., Wong, H., Lee, G., Leete, T. H., Ronk, M., Shively, J. E., Sternby, B., Borgström, B., Ameis, D. and Scholtz, M. C. (1991) J. Lipid. Res. 32, 267-276.  
         [0085]    Wang, C-S, and Hartsuck, J. A. (1993) Biochim. Biphys Acta 1166, 1-19.  
         [0086]    Deposit Of Microorganisms  
         [0087]    The following plasmids, transformed into  Pichia pastoris  cultures, have been deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, Scotland, UK. The date of deposit is May 2, 1995.  
                                                   Strain[plasmid]   NCIMB No.                           PPF-1[pARC 5771]   40721           GS115[pARC 5799]   40723           GS115[pARC 5797]   40722                      
 
         [0088]    [0088]                                                                                               TABLE 1                           Enzyme activity in the culture supernatants of  Pichia pastoris         transformants.                Enzyme activity in mg/L equivalent of native BSSL                PPF-   GS115           Hours after   1[pARC 5771]   [pARC 5799]   GS115[pARC 5797]            induction   No.39   No.86   No.9   No.21   No.3                    24   0.254   0.135   1.53   1.72   0.37       48   2.69   3.12   17.28   34.70   40.9       72   3.96   8.25   37.37   50.60   44.9       96   11.26   13.60   26.34   50.60   35.6       120   8.42   13.13   13.60   22.30   17.8                    
         [0089]    [0089] 
     
       
       
         1 
         
           
             
4 
 
           
           
             
               2428 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               cDNA to mRNA  
             
             NO  
             NO  
             
               Homo sapiens  
               mammary gland  
             
             
               CDS  
                82..2319  
                /product= “bile-salt-stimulated 
               lipase”
 
             
             
               exon  
                985..1173 

 
             
             
               exon  
                1174..1377 

 
             
             
               exon  
                1378..1575 

 
             
             
               exon  
                1576..2415 

 
             
             
               mat_peptide  
                151..2316 

 
             
             
               polyA_signal  
                2397..2402 

 
             
             
               repeat_region  
                1756..2283 

 
             
             
               5′UTR  
                1..81 

 
             
             
               repeat_unit  
                1756..1788 

 
             
             
               repeat_unit  
                1789..1821 

 
             
             
               repeat_unit  
                1822..1854 

 
             
             
               repeat_unit  
                1855..1887 

 
             
             
               repeat_unit  
                1888..1920 

 
             
             
               repeat_unit  
                1921..1953 

 
             
             
               repeat_unit  
                1954..1986 

 
             
             
               repeat_unit  
                1987..2019 

 
             
             
               repeat_unit  
                2020..2052 

 
             
             
               repeat_unit  
                2053..2085 

 
             
             
               repeat_unit  
                2086..2118 

 
             
             
               repeat_unit  
                2119..2151 

 
             
             
               repeat_unit  
                2152..2184 

 
             
             
               repeat_unit  
                2185..2217 

 
             
             
               repeat_unit  
                2218..2250 

 
             
             
               repeat_unit  
                2251..2283 

 
             
             
               
                 
                   Jeanette 
               Blackberg, Lars 
               Carlsson, Peter 
               Enerback, Sven 
               Hernell, Olle 
               Bjursell, Gunnar  
                   Nilsson 
                 
               
                cDNA cloning of human-milk 
               bile-salt-stimulated lipase and evidence for its 
               identity to pancreatic carboxylic ester hydrolase  
                Eur. J. Biochem.  
                192  
                543-550  
               
 Sept.-1990
 
             
             1 

ACCTTCTGTA TCAGTTAAGT GTCAAGATGG AAGGAACAGC AGTCTCAAGA TAATGCAAAG     60 

AGTTTATTCA TCCAGAGGCT G ATG CTC ACC ATG GGG CGC CTG CAA CTG GTT      111 
                        Met Leu Thr Met Gly Arg Leu Gln Leu Val 
                        -23         -20                 -15 

GTG TTG GGC CTC ACC TGC TGC TGG GCA GTG GCG AGT GCC GCG AAG CTG      159 
Val Leu Gly Leu Thr Cys Cys Trp Ala Val Ala Ser Ala Ala Lys Leu 
            -10                  -5                   1 

GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA GGC GTC AAT AAG AAG      207 
Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val Asn Lys Lys 
      5                  10                  15 

CTC GGC CTC CTG GGT GAC TCT GTG GAC ATC TTC AAG GGC ATC CCC TTC      255 
Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly Ile Pro Phe 
 20                  25                  30                  35 

GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG CCA CAT CCT GGC TGG      303 
Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His Pro Gly Trp 
                 40                  45                  50 

CAA GGG ACC CTG AAG GCC AAG AAC TTC AAG AAG AGA TGC CTG CAG GCC      351 
Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala 
             55                  60                  65 

ACC ATC ACC CAG GAC AGC ACC TAC GGG GAT GAA GAC TGC CTG TAC CTC      399 
Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu 
         70                  75                  80 

AAC ATT TGG GTG CCC CAG GGC AGG AAG CAA GTC TCC CGG GAC CTG CCC      447 
Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg Asp Leu Pro 
     85                  90                  95 

GTT ATG ATC TGG ATC TAT GGA GGC GCC TTC CTC ATG GGG TCC GGC CAT      495 
Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly Ser Gly His 
100                 105                 110                 115 

GGG GCC AAC TTC CTC AAC AAC TAC CTG TAT GAC GGC GAG GAG ATC GCC      543 
Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala 
                120                 125                 130 

ACA CGC GGA AAC GTC ATC GTG GTC ACC TTC AAC TAC CGT GTC GGC CCC      591 
Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg Val Gly Pro 
            135                 140                 145 

CTT GGG TTC CTC AGC ACT GGG GAC GCC AAT CTG CCA GGT AAC TAT GGC      639 
Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly 
        150                 155                 160 

CTT CGG GAT CAG CAC ATG GCC ATT GCT TGG GTG AAG AGG AAT ATC GCG      687 
Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg Asn Ile Ala 
    165                 170                 175 

GCC TTC GGG GGG GAC CCC AAC AAC ATC ACG CTC TTC GGG GAG TCT GCT      735 
Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala 
180                 185                 190                 195 

GGA GGT GCC AGC GTC TCT CTG CAG ACC CTC TCC CCC TAC AAC AAG GGC      783 
Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly 
                200                 205                 210 

CTC ATC CGG CGA GCC ATC AGC CAG AGC GGC GTG GCC CTG AGT CCC TGG      831 
Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu Ser Pro Trp 
            215                 220                 225 

GTC ATC CAG AAA AAC CCA CTC TTC TGG GCC AAA AAG GTG GCT GAG AAG      879 
Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val Ala Glu Lys 
        230                 235                 240 

GTG GGT TGC CCT GTG GGT GAT GCC GCC AGG ATG GCC CAG TGT CTG AAG      927 
Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln Cys Leu Lys 
    245                 250                 255 

GTT ACT GAT CCC CGA GCC CTG ACG CTG GCC TAT AAG GTG CCG CTG GCA      975 
Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala 
260                 265                 270                 275 

GGC CTG GAG TAC CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT GTC ATT     1023 
Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val Pro Val Ile 
                280                 285                 290 

GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC AAC GCC     1071 
Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala 
            295                 300                 305 

GCC GAC ATC GAC TAT ATA GCA GGC ACC AAC AAC ATG GAC GGC CAC ATC     1119 
Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp Gly His Ile 
        310                 315                 320 

TTC GCC AGC ATC GAC ATG CCT GCC ATC AAC AAG GGC AAC AAG AAA GTC     1167 
Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn Lys Lys Val 
    325                 330                 335 

ACG GAG GAG GAC TTC TAC AAG CTG GTC AGT GAG TTC ACA ATC ACC AAG     1215 
Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr Ile Thr Lys 
340                 345                 350                 355 

GGG CTC AGA GGC GCC AAG ACG ACC TTT GAT GTC TAC ACC GAG TCC TGG     1263 
Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp 
                360                 365                 370 

GCC CAG GAC CCA TCC CAG GAG AAT AAG AAG AAG ACT GTG GTG GAC TTT     1311 
Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val Val Asp Phe 
            375                 380                 385 

GAG ACC GAT GTC CTC TTC CTG GTG CCC ACC GAG ATT GCC CTA GCC CAG     1359 
Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala Leu Ala Gln 
        390                 395                 400 

CAC AGA GCC AAT GCC AAG AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC     1407 
His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser 
    405                 410                 415 

CAT CCC TCT CGG ATG CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT     1455 
His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His 
420                 425                 430                 435 

GCA GAT GAC ATT CAG TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG     1503 
Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr 
                440                 445                 450 

GGC TAC CGG CCC CAA GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC     1551 
Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr 
            455                 460                 465 

TGG ACC AAC TTT GCC AAA ACA GGG GAC CCC AAC ATG GGC GAC TCG GCT     1599 
Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly Asp Ser Ala 
        470                 475                 480 

GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG GAA AAC AGC GGC TAC CTG     1647 
Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu 
    485                 490                 495 

GAG ATC ACC AAG AAG ATG GGC AGC AGC TCC ATG AAG CGG AGC CTG AGA     1695 
Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg Ser Leu Arg 
500                 505                 510                 515 

ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC TAT CTG GCG CTG CCC ACA     1743 
Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr 
                520                 525                 530 

GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC CCC ACA GGG GAC TCC GAG     1791 
Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu 
            535                 540                 545 

GCC ACT CCC GTG CCC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG     1839 
Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro 
        550                 555                 560 

CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC     1887 
Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser 
    565                 570                 575 

GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG     1935 
Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 
580                 585                 590                 595 

CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC     1983 
Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp 
                600                 605                 610 

TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC     2031 
Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro 
            615                 620                 625 

GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC CCC GTG CCG CCC ACG GGT     2079 
Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly 
        630                 635                 640 

GAC GCC GGG CCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC     2127 
Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro 
    645                 650                 655 

CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG ACC CCC ACG     2175 
Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Thr Pro Thr 
660                 665                 670                 675 

GGT GAC TCC GAG ACC GCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC     2223 
Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly Ala 
                680                 685                 690 

CCC CCT GTG CCC CCC ACG GGT GAC TCT GAG GCT GCC CCT GTG CCC CCC     2271 
Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro Val Pro Pro 
            695                 700                 705 

ACA GAT GAC TCC AAG GAA GCT CAG ATG CCT GCA GTC ATT AGG TTT TAG     2319 
Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile Arg Phe  * 
        710                 715                 720 

CGTCCCATGA GCCTTGGTAT CAAGAGGCCA CAAGAGTGGG ACCCCAGGGG CTCCCCTCCC   2379 

ATCTTGAGCT CTTCCTGAAT AAAGCCTCAT ACCCCTAAAA AAAAAAAAA               2428 

 
           
           
             
               745 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             2 

Met Leu Thr Met Gly Arg Leu Gln Leu Val Val Leu Gly Leu Thr Cys 
-23         -20                 -15                 -10 

Cys Trp Ala Val Ala Ser Ala Ala Lys Leu Gly Ala Val Tyr Thr Glu 
         -5                   1               5 

Gly Gly Phe Val Glu Gly Val Asn Lys Lys Leu Gly Leu Leu Gly Asp 
 10                  15                  20                  25 

Ser Val Asp Ile Phe Lys Gly Ile Pro Phe Ala Ala Pro Thr Lys Ala 
                 30                  35                  40 

Leu Glu Asn Pro Gln Pro His Pro Gly Trp Gln Gly Thr Leu Lys Ala 
             45                  50                  55 

Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala Thr Ile Thr Gln Asp Ser 
         60                  65                  70 

Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu Asn Ile Trp Val Pro Gln 
     75                  80                  85 

Gly Arg Lys Gln Val Ser Arg Asp Leu Pro Val Met Ile Trp Ile Tyr 
 90                  95                 100                 105 

Gly Gly Ala Phe Leu Met Gly Ser Gly His Gly Ala Asn Phe Leu Asn 
                110                 115                 120 

Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala Thr Arg Gly Asn Val Ile 
            125                 130                 135 

Val Val Thr Phe Asn Tyr Arg Val Gly Pro Leu Gly Phe Leu Ser Thr 
        140                 145                 150 

Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly Leu Arg Asp Gln His Met 
    155                 160                 165 

Ala Ile Ala Trp Val Lys Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro 
170                 175                 180                 185 

Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala Gly Gly Ala Ser Val Ser 
                190                 195                 200 

Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly Leu Ile Arg Arg Ala Ile 
            205                 210                 215 

Ser Gln Ser Gly Val Ala Leu Ser Pro Trp Val Ile Gln Lys Asn Pro 
        220                 225                 230 

Leu Phe Trp Ala Lys Lys Val Ala Glu Lys Val Gly Cys Pro Val Gly 
    235                 240                 245 

Asp Ala Ala Arg Met Ala Gln Cys Leu Lys Val Thr Asp Pro Arg Ala 
250                 255                 260                 265 

Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala Gly Leu Glu Tyr Pro Met 
                270                 275                 280 

Leu His Tyr Val Gly Phe Val Pro Val Ile Asp Gly Asp Phe Ile Pro 
            285                 290                 295 

Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala Ala Asp Ile Asp Tyr Ile 
        300                 305                 310 

Ala Gly Thr Asn Asn Met Asp Gly His Ile Phe Ala Ser Ile Asp Met 
    315                 320                 325 

Pro Ala Ile Asn Lys Gly Asn Lys Lys Val Thr Glu Glu Asp Phe Tyr 
330                 335                 340                 345 

Lys Leu Val Ser Glu Phe Thr Ile Thr Lys Gly Leu Arg Gly Ala Lys 
                350                 355                 360 

Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp Ala Gln Asp Pro Ser Gln 
            365                 370                 375 

Glu Asn Lys Lys Lys Thr Val Val Asp Phe Glu Thr Asp Val Leu Phe 
        380                 385                 390 

Leu Val Pro Thr Glu Ile Ala Leu Ala Gln His Arg Ala Asn Ala Lys 
    395                 400                 405 

Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met Pro 
410                 415                 420                 425 

Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gln Tyr 
                430                 435                 440 

Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gln Asp 
            445                 450                 455 

Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn Phe Ala Lys 
        460                 465                 470 

Thr Gly Asp Pro Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu 
    475                 480                 485 

Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu Glu Ile Thr Lys Lys Met 
490                 495                 500                 505 

Gly Ser Ser Ser Met Lys Arg Ser Leu Arg Thr Asn Phe Leu Arg Tyr 
                510                 515                 520 

Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr Val Thr Asp Gln Glu Ala 
            525                 530                 535 

Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Thr Pro Val Pro Pro 
        540                 545                 550 

Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly 
    555                 560                 565 

Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro 
570                 575                 580                 585 

Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser 
                590                 595                 600 

Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 
            605                 610                 615 

Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp 
        620                 625                 630 

Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ala Gly Pro Pro Pro 
    635                 640                 645 

Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly 
650                 655                 660                 665 

Asp Ser Gly Ala Pro Pro Val Thr Pro Thr Gly Asp Ser Glu Thr Ala 
                670                 675                 680 

Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr 
            685                 690                 695 

Gly Asp Ser Glu Ala Ala Pro Val Pro Pro Thr Asp Asp Ser Lys Glu 
        700                 705                 710 

Ala Gln Met Pro Ala Val Ile Arg Phe 
    715                 720 

 
           
           
             
               722 amino acids  
               amino acid  
                 
               linear  
             
             
               protein  
             
             NO  
             
               Homo sapiens  
               Mammary gland  
             
             3 

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val 
1               5                   10                  15 

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly 
            20                  25                  30 

Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His 
        35                  40                  45 

Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys 
    50                  55                  60 

Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys 
65                  70                  75                  80 

Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg 
                85                  90                  95 

Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly 
            100                 105                 110 

Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu 
        115                 120                 125 

Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg 
    130                 135                 140 

Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly 
145                 150                 155                 160 

Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg 
                165                 170                 175 

Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly 
            180                 185                 190 

Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr 
        195                 200                 205 

Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu 
    210                 215                 220 

Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val 
225                 230                 235                 240 

Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln 
                245                 250                 255 

Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val 
            260                 265                 270 

Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val 
        275                 280                 285 

Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr 
    290                 295                 300 

Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp 
305                 310                 315                 320 

Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn 
                325                 330                 335 

Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr 
            340                 345                 350 

Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr 
        355                 360                 365 

Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val 
    370                 375                 380 

Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala 
385                 390                 395                 400 

Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr 
                405                 410                 415 

Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly 
            420                 425                 430 

Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala 
        435                 440                 445 

Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met 
    450                 455                 460 

Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly 
465                 470                 475                 480 

Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser 
                485                 490                 495 

Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg 
            500                 505                 510 

Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala 
        515                 520                 525 

Leu Pro Thr Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly 
    530                 535                 540 

Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala 
545                 550                 555                 560 

Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr 
                565                 570                 575 

Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala 
            580                 585                 590 

Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro 
        595                 600                 605 

Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly 
    610                 615                 620 

Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro 
625                 630                 635                 640 

Pro Thr Gly Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser 
                645                 650                 655 

Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 
            660                 665                 670 

Thr Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp 
        675                 680                 685 

Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro 
    690                 695                 700 

Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile 
705                 710                 715                 720 

Arg Phe 

 
           
           
             
               568 amino acids  
               amino acid  
                 
               linear  
             
             
               protein  
             
             NO  
             
               Homo sapiens  
               Mammary gland  
             
             
               Peptide  
                1..568  
                /label= Variant_C 
 
             
             
               
                 
                   Lennart 
               Blackberg, Lars 
               Edlund, Michael 
               Lundberg, Lennart 
               Stromqvist, Mats 
               Hernell, Olle  
                   Hansson 
                 
               
                Recombinant Human Milk Bile Salt-stimulated 
               Lipase  
                J. Biol. Chem.  
                268  
                35  
                26692-26698  
               
 Dec. 15-1993
 
             
             4 

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val 
1               5                   10                  15 

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly 
            20                  25                  30 

Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His 
        35                  40                  45 

Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys 
    50                  55                  60 

Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys 
65                  70                  75                  80 

Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg 
                85                  90                  95 

Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly 
            100                 105                 110 

Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu 
        115                 120                 125 

Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg 
    130                 135                 140 

Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly 
145                 150                 155                 160 

Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg 
                165                 170                 175 

Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly 
            180                 185                 190 

Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr 
        195                 200                 205 

Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu 
    210                 215                 220 

Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val 
225                 230                 235                 240 

Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln 
                245                 250                 255 

Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val 
            260                 265                 270 

Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val 
        275                 280                 285 

Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr 
    290                 295                 300 

Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp 
305                 310                 315                 320 

Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn 
                325                 330                 335 

Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr 
            340                 345                 350 

Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr 
        355                 360                 365 

Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val 
    370                 375                 380 

Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala 
385                 390                 395                 400 

Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr 
                405                 410                 415 

Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly 
            420                 425                 430 

Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala 
        435                 440                 445 

Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met 
    450                 455                 460 

Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly 
465                 470                 475                 480 

Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser 
                485                 490                 495 

Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg 
            500                 505                 510 

Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala 
        515                 520                 525 

Leu Pro Thr Val Thr Asp Gln Gly Ala Pro Pro Val Pro Pro Thr Gly 
    530                 535                 540 

Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Lys Glu Ala 
545                 550                 555                 560 

Gln Met Pro Ala Val Ile Arg Phe 
                565