MXPA97006853A - A dna molecule for expression of bile salt-stimulated lipase (bssl) - Google Patents

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. The DNA molecule comprising:(a) a region coding for a polypeptide which is human BSSL or a biologically active variant thereof;(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 (c) operably-linked to said coding regions defined in (a) and (b), the methanol oxidase promoter of Pichia pastoris or a functionally equivalent promoter.

Description

A MOLECULE. OF DNA FOR THE EXPRESSION OF LIPASA STIMULATED BY BILIARY SALTS TECHNICAL FIELD The invention relates to DNA molecules, recombinant vectors and cell cultures for use in methods for the expression of lipase stimulated by bile salts (BSSL) in the methylotrophic yeast Pichia pastoris.

BACKGROUND OF THE INVENTION Lipase stimulated by bile salts (BSSL; EC 3.1.1.1) (for a review see ang &Hartsuck, 1993) contributes to most of the lipolytic activity of human milk. A major feature of this lipase is that it requires the primary bile salts for activity against the emulsified long chain triacylglycerols. BSSL has been found only in human, gorilla, cat and dog milk (Hernell et al., 1989). The BSSL has been attributed a critical role in the digestion of milk lipids in the intestine of lactating infants (Fredrikzon et al., 1978). BSSL is synthesized in humans in the lactating mammary gland and secreted with milk (Bláckberg et al., 1987). This REF: 25559 contributes approximately 1% of the total milk protein (Bláckberg &Hernell, 1981). It has been suggested that BSSL is the main limiting factor for the rate of fat absorption and the subsequent growth of infants, particularly premature infants, who are deficient in their own production of BSSL, and that supplementing formulas with purified enzyme improves significantly the digestion and growth of these infants (US 4,944,944; Oklahoma Medical Research Foundation). Thus, it is clinically important in the preparation of infant formulas that contain relatively high percentages of triglycerides and that are based on sources of protein, plants or non-human milk, since the infants fed with these formulas are unable to digest fat in the absence of the BSSL * S- ~ added. The cDNA structures have been characterized for both the BSSL of the milk and the carboxylic hydrolase ester (CEH) of the pancreas (Baba et al., 1991; Hui et al.

Kissel, 1991; Nilsson et al., 1991; Reue et al., 1991) and it has been concluded that the enzyme of milk and the enzyme of the pancreas are products of the same gene, the CEL gene. The sequence of the cDNA (SEQ ID NO: 1) of the CEL gene is described in US Patent 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 23 amino acid signal sequence, is shown as SEQ. ID. NO: 2 in the Sequence Listing, while the native protein sequence of 722 amino acids is shown as SEQ. ID. NO: 3. The C-terminal region of the protein contains 16 repeats of 11 residual amino acids each, followed by an extension of 11 conserved amino acids. The native protein is highly glycosylated and a wide range of observed molecular weights have been reported. This can probably be explained by varying the degree of glycosylation (Abouakil et al., 1988). The N-terminal half of the protein is homologous to acetyl choline esterase and some other esterases (Nilsson et al., 1990). Recombinant BSSL can be produced by expression in a suitable host such as E. coli, Saccharomyces cerevisiae, or mammalian cell lineages. For the scaling of a BSSL expression system to make commercially viable production costs, the use of heterologous expression systems could be contemplated. As mentioned above, the human BSSL has 16 repeats of 11 amino acids at the C-terminal end. To determine the biological significance of this repeated region, several mutants of the human BSSL have been constructed that lack part or all of the repeated regions (Hansson et al., 1993). The variant BSSL-C (SEQ ID NO: 4), for example, has deleted the residual amino acids 536 to 568 and the residual amino acids 591 to 711. Expression studies, using as host the mammalian cell line C127 and the bovine papilloma virus expression vector, showed that different variants can be expressed in active forms (Hansson et al., 1993). From the expression studies it was also concluded that the rich repeats in Proline in human BSSL are not essential for the catalytic activity or activation of BSSL by bile salts. However, the production of BSSL or its mutants in a mammalian expression system could be very expensive for routine therapeutic use. A eukaryotic system such as a yeast can provide significant advantages, compared to the use of prokaryotic systems, for the production of certain polypeptides encoded by the recombinant DNA. For example, yeast can generally grow at densities cells greater than bacteria and may be able to glycosylate the expressed polypeptides, wherein such glycosylation is important for biological activity. However, the use of the yeast Saccharomyces cerevisiae as a host organism often leads to poor levels of Expression and a poor secretion of the recombinant protein (Cregg et al., 1987). The maximum levels of heterologous proteins in S. cerevisiae are in the 5% region of the total cellular protein (Kingsman et al., 1985). A further disadvantage of using Saccharomyces cerevisiae as a host is that the recombinant proteins tend to be overglycosylated, which could affect the activity of the glycosylated mammalian proteins. Pichia pastoris is a methylotrophic yeast, which can grow on methanol as the sole source of carbon and energy since it contains a highly regulated methanol utilization pathway (Ellis et al., 1985). The P. Pastoris is also manageable for efficient high-density cell fermentation technology. Therefore, recombinant DNA technology and efficient methods of yeast transformation have made it possible to develop P. pastoris as a host for the expression of heterologous protein in large quantities, with an expression system based on the methanol promoter. oxidase (Cregg et al., 1987). The use of Pichia pastoris is known in the art as a host for the expression of, for example, the following heterologous proteins: human tumor necrosis factor (EP-A-0263311); Bordetella pertactin antigens (WO 91/15571); surface antigen of hepatitis B (Cregg et al., 1987); human lysozyme protein (WO 92/04441); Aprotinin (WO 92/01048). However, the successful expression of a heterologous protein in active, soluble and secreted form depends on several factors, for example, correct choice of the signal peptide, proper construction of the fusion junction between the signal peptide and the mature protein, conditions of growth, etc.

PURPOSES OF THE INVENTION The purpose of the invention is to overcome the disadvantages mentioned above with the above systems and to provide a method for the production of human BSSL at low cost and with a performance comparable to, or superior to, production in other organisms. This purpose has been achieved by providing methods for the expression of BSSL in Pichia pastoris cells. By means of the invention in this way it has been shown that the human BSSL and the variant BSSL-C can be expressed in the active secreted form of P. pastoris. The native signal peptide, as well as the heterologous signal peptide derived from invertase protein of S. cerevisiae, have been used to translocate the mature protein in the culture medium as an appropriately processed form.

BRIEF DESCRIPTION OF THE INVENTION * In a first aspect, the invention provides a DNA molecule comprising: (a) a region encoding a polypeptide, which is the human BSSL or a biologically active variant thereof; (b) attached to the 5 'end of the coding region 'SP- for the polypeptide, a region that codes for a peptide signal capable of directing the secretion of the polypeptide of Pichia pastoris cells transformed with such a DNA molecule; and (c) operably linked to the coding regions defined in (a) and (b), the promoter of the methanol oxidase from Pichia pastoris or a functionally equivalent promoter.

The term "biologically active variant" of the BSSL should be understood as a polypeptide having BSSL activity comprising part of the amino acid sequence shown as SEQ. ID. NO: 3 in the Sequence Listing. The term "polypeptide having BSSL activity" in this context should be understood as a polypeptide comprising the following properties: (a) is suitable for oral administration; (b) is activated by specific bile salts; and (c) acts as a non-specific lipase in the * context of the small intestine, that is to say that it is able to hydrolyze lipids relatively, independently of its chemical structure and physical state (emulsified, micellar, soluble). Such a variant of the BSSL may for example be a variant comprising less than 16 repeated units, whereby a "repeated unit" shall be understood as a * repeating unit of 11 amino acids, encoded by a nucleotide sequence indicated as a "repeating unit" under the heading "(ix) CHARACTERISTICS" in "INFORMATION FOR SEQ ID NO: 1" in the Sequence Listing. In particular, the variant of the BSSL may be the variant BSSL-C, where amino acids 536 to 568 and 591 to 711 have been deleted (SEQ ID NO: 4 in the Sequence Listing). Accordingly, the DNA molecule according to the invention is preferably a DNA molecule that codes for BSSL (SEQ ID NO: 3) or BSSL-C (SEQ ID NO: 4). However, the DNA molecules according to the The invention is not strictly limited to the DNA molecules encoding the polypeptides with amino acid sequences identical to SEQ. ID. NO: 3 or 4 in the Sequence Listing. In addition, the invention encompasses the DNA molecules that code for polypeptides that contain modifications as well as substitutions, small -f deletions, insertions or inversions, polypeptides which nevertheless have substantially the biological activities of the BSSL. Included in the invention are therefore the DNA molecules encoding the BSSL variants set forth above and also the DNA molecules encoding the polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the sequence # of amino acids shown as SEQ. ID. NO: 3 or 4 in the Listing of Sequences. The signal peptide referred to above may 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 an invertase signal peptide of Saccharomyces cerevisiae. In a further aspect, the invention provides a vector comprising a DNA molecule as defined above. Preferably, such vector is a duplicatable expression vector which contains and is capable of mediating the expression, in a Pichia genus, of a DNA sequence encoding the human BSSL or a biologically active variant thereof. Such a vector may, for example, be the plasmid vector pARC 5771 (NCIMB 40721), pARC 5799 (NCIMB 40723) or pARC 5797 (NCIMB 40722). In another aspect, the invention provides a culture of host cells comprising cells of the genus Pichia transformed with a DNA molecule or a vector as defined above. Preferably, the host cells are Pichia pastoris strains cells * such as PPF-1 or GS115. The cell culture can, for example, be the PPF-I culture [pARC 5771] (NCIMB 40721), GS115 [pARC 5799] (NCIMB 40723) or GSll5 [pARC 5797] (NCIMB 40722). In still another aspect, the invention provides a process for the production of a polypeptide which is the Human BSSL, or a biologically active variant thereof, which comprises culturing host cells according to the invention under conditions whereby such polypeptide is secreted into the culture medium, and recovering the polypeptide from the host medium. culture. -f EXAMPLES OF THE INVENTION EXAMPLE 1: Expression of the BSSL in P ± ch ± a pastoris PPF-1 1. 1. Construction of pARC 0770 The cDNA sequence (SEQ ID NO: 1) which codes for the BSSL protein, which includes the native signal peptide (referred to later as NSP), was cloned into pTZ19R (Pharmacia) with an EcoRI-SacI fragment. The cloning of the NSP-BSSL cDNA into the expression vector pSC 231 of 5. cerevisiae (obtained from Professor L. Prakash, University of Rochester, NY, USA), which allows a vector of expression of yeast under copy number, wherein the expression is under the control of the constitutive ADHl promoter, was achieved in two steps. Initially the NSP-BSSL cDNA was cloned into pYES 2.0 (Invitrogen, USA) as a £ coRI-Sp? I of pTZ19R-SP-BSSL. The 89 base pairs in excess between the £ coRI and Ncol at the beginning of the sequence encoding the signal peptide were removed creating a fusion . EcoRI / NcoI (89) and regenerating an EcoRl site. The resulting clone pARC 0770 contained an ATG codon, originally encoded within the Ncol site which was immediately followed by the EcoRI site regenerated in frame with the remaining βSS-BSSL sequence. -f 1.2. Construction of plasmid pARC 5771 To construct a suitable expression vector for BSSL expression, the cDNA fragment coding for the BSSL protein together with its native signal peptide was cloned with the expression vector pDM 148 of P. pastoris. The vector pDM 148 (received from Dr. S. Subramani, UCSD) was constructed as follows: the untranslated region upstream (5'-UTR) and the untranslated region downstream (3'-UTR) of the methanol oxidase gene (MOX1) were isolated by PCR and cascaded in the multiple cloning sequence (MCS) of the pSK + vector of E. coli (available from Stratagene, USA). For the proper selection of transformants of putative P. pastoris, a DNA sequence encoding the ARG4 gene of 5. cerevisiae was inserted along with its own promoter sequence between the 5'- and 3 '-UTR in pSK-. The resulting construct pDM148 has the following characteristics: in the MCS region of the pSK the 5 '-UTR of MOX, the genomic sequence of S. cerevisiae ARG4 and the 3 '-UTR of MOX. Between the 5 * -UTR of MOX and the genomic sequence of ARG4 a series of unique restriction sites was placed (Sa II, Clal, EcóRI, PstI, Smal and Bamñl) where any sequence that codes for a heterologous protein can be cloned for expression under the control of the MOX promoter in P. pastoris. To facilitate the integration of this expression cassette into the M0X1 site in the P. pastoris chromosome, the expression cassette can be excised from the rest of the pSK vector "by digestion with the restriction enzyme Notl. MOX1 of P. pastoris cloned in the pDM 148 was approximately 500 bp in length while the 3 '-UTR of MOX1 of P. pastoris cloned in the pDM 148 was W approximately 1000 bp in length. To insert the AD? C sequence of? SP-BSSL, between the 5'-UTR of MOX1 and the sequence coding for S. cerevisiae ARG4 in pDM 148, the AD? C insert (SP-BSSL) was isolated of pARC 0770 by digestion with £ coRI and Ba HI (AD fragment of approximately 2.2 kb) and cloned between the coRI sites and BamHI in pDM 148.) The resulting construct pARC 5771 (? CIMB 40721) contained the 5'-UTR of MOXl from P. pastoris followed by the sequence that sequence encoding? SP-BSSL followed by the sequence of the ARG4 gene of S. cerevisiae and the 3 '-UTR of the MOXl gene of P. pastoris while the entire segment of AD? of the 5 '-UTR of MOXl at 3 '-UTR of MOXl was cloned into the MCS of pSKA 1.3. Transformation of BSSL in P. pastoris host PPF-1 7 For the expression of the BSSL in P. pastoris host PPF-1 (his4, arg4; received from Phillips Petroleum Co.), the plasmid pARC 5771 was digested with Notl and all the digested mixture (10 μg of total AD?) Was used. to transform to PPF-1. The transformation protocol followed was essentially the seroplast method of yeast described by Cregg et al. (1987). The transformants were generated on minimal medium without arginine so that the Arg + colonies could be selected. The regeneration of the upper agar containing the transformants was raised and homogenized in water and the yeast cells were cultured at approximately 250 colonies per plate on plates minimum glucose without arginine. The mutant colonies were identified by growing duplicates in minimal methanol plates. Approximately 15% of all transformants became Muts phenotype (slow growth in methanol). 1.4. Selection of transformants expressing BSSL To select a large number of transformants rapidly for the expression of lipase a plaque assay method for lipase was developed. The procedure for The preparation of these lacquers was as follows: to a 2% agarose solution (final), a 10 x Na-cholate solution in water was added to a final concentration of 1%. The lipid substrate tributine was added to the mixture at a final concentration of 1% (v / v). To support the growth of the transformants, the mixture is further supplemented with 0.25% yeast nitrogen base (final) and 0.5% methanol (final). The ingredients were mixed properly and poured into plates to a thickness of 3-5 mm. Once the mixture solidified, the transformants were dispersed on the plates and the plates were further incubated at + 37 ° C for 12 h. The clones that produce lipase showed a clear halo around the clone. In a typical experiment 7 of a total of 93 transformants were identified as transformants that produce BSSL. Two clones (Nos. 39 and 86) that produced the largest haloes around the dispersed colony were extracted for further characterization. 1. 5. Expression of the BSSL of PPF-l [pARC 5771] The two transformants Nos. 39 and 86 described in Section 1.4 was extracted and made to grow in liquid medium BMGY (1% yeast extract, 2% bactopeptone, 1.34% yeast-free nitrogen base, 100 mM KP04 buffer, pH 6.0, 400 μg / 1 biotin, and 2% glycerol) for 24 hours at 30 ° C until the cultures reached an Aeoo close to 40. The cultures were sedimented and resuspended in BMMY media (2% glycerol replaced by 0.5% methanol in BMGY) to A6oo = 300. Induced cultures were incubated at 30 ° C. ° C with shaking for 120 h. Culture supernatants were extracted at different times for the analysis of BSSL expression by enzymatic activity assay, SDS-PAGE analysis and western spotting. 10 1.6. Detection of enzymatic activity of BSSL in culture supernatants of clones Nos. 39 and 86 To determine the enzymatic activity in the supernatant of cell-free culture of induced tfL cultures Nos. 39 and 86 as described in Section 1.5, cultures were centrifuged and 2 μl of cell-free supernatant was assayed for BSSL enzymatic activity according to the method described by Hernell and 20 Olivecrona (1974). As shown in Table 1, it was found that both cultures contain enzymatic activity of BSSL with the maximum activity at 96 h hours after induction. 1. 7. Western blot analysis of the culture supernatants of the PPF-1 transformants: pARC 5771 (Nos. 39 and 86) To determine the presence of the recombinant BSSL in the culture supernatants Nos. 39 and 86 of the PPF-1 transformants [pARC 5771], the cultures were grown and induced as described in Section 1.5. Cultures were extracted at different times after 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 the BSSL in Pichia pastoris GS115 2.1. Construction of the pARC 5799 Since the 5 '-MOX UTR and the 3' -MOX UTR were not properly defined and since the vector pDM 148 lacks any other suitable marker (e.g., a resistance gene G418) to verify the number of copies of the integrated BSSL on the Pichia chromosome, the cDNA insert of the native BSSL together with its signal peptide was cloned into another expression vector of P. pastoris, pHIL D4. He Integrated pHIL D4 plasmid was obtained from Phillips Petroleum Company. The plasmid contained 5'-MOXl, a segment of approximately 1000 bp of the alcohol oxidase promoter and a single coRI cloning site. It also contained approximately 250 bp of the 3 '-MOXl region containing the termination sequence of the alcohol oxidase, followed by the EcoRI site. The "termination" region was followed by the HIS4 gene of P. pastoris histidinol dehydrogenase contained in a 2.8 kb fragment to complement the defective HIS4 gene in host GS115 (see below). A 650 bp region containing the 3 '-MOXl DNA was fused to the 3' end of the HIS4 gene, which together with the 5 '-MOXl region was necessary for site-directed integration. A bacterial kanamycin resistance gene from pUC-4K (PL-Biochemicals) was inserted into the unique Nael site between HIS4 and the 3 * -MOXl region at 3? of the HIS4 gene. To clone the fragment of AD? coding for? SP-BSSL at the site of the single coIL of pHIL D4, a double-stranded oligo-linker having a BamHI-EcoRI cleaved position was ligated to plasmid pARC 5771 digested with BamHI and the entire sequence coding for? SP-BSSL it was extracted as a 2.2 kb £ coRI fragment. This fragment was cloned into the £ csRI site of pHIL D-4 and the correctly oriented plasmid was designated pARC 5799 (? CIMB 40723). 2. 2. Transformation of pARC 5799 To facilitate the integration of the sequence coding for NSP-BSSL into the genomic site of MOXl, P. pastoris of plasmid pARC 5799 was digested with BglII and used for the 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 the His prototype. The transformants are extracted after cultivation by serial dilution of the regenerated upper agar and tested directly by the lipase plate assay as described in Section 1.4. Two transformant clones (Nos. 9 and 21) were extracted on the basis of the size of the halo on the lipase assay plate and also verified for the expression of BSSL. It was found that the clones were MutA # 2. 3. Determination of the enzymatic activity of the BSSL in the culture supernatants of the GS115 transformants [pARC 5799] Nos. 9 and 21 The two transformed clones Nos. 9 and 21 of GS115 [pARC 5799] were grown essentially following the protocol described in Section 1.5. Culture supernatants were tested at different times after induction for the BSSL enzymatic activity as described in Section 1.6. As shown in Table 1, it was found that both culture supernatants contained enzymatic activity of BSSL and enzyme activity was greater 72 h after induction. Both clones showed a higher expression of BSSL compared to clones of PPF-l [pARC 5771]. * 10 2.4. SDS-PAGE and western blot analysis of culture supernatants of GSU5 transformants [pARC 5799] Nos. 9 and 21 Culture supernatants harvested at different times, as described in Section 2.3, were subjected to SDS-PAGE and western blot analysis. From the SDS-PAGE profile it was estimated that approximately 60-75% of the total protein present in the culture supernatants of the induced cultures was BSSL. The molecular weight of the The protein was approximately 116 kDa. Western blotting data also confirmed that the majority of the protein present in the culture supernatant was BSSL. The protein apparently had the same molecular weight as the Native BSSL. 25 EXAMPLE 3: Scaling of the BSSL expression 3. 1. Scaling of the BSSL expression of the GS115 transformed clone [pARC 5799] (No. 21) A B. Braun fermenter of 23 1 capacity was used. Five liters of medium containing, 1% YE, 2% Peptone, 1.34 YNB and 4% w / v glycerol * sterilized in an autoclave at 121 ° C for 30 minutes and added biotin (final concentration of 400 μg / L) during inoculation after sterilization by filtering. For the inoculum, GS115 standard [pARC 5799] (No. 21) glycerol was inoculated into a synthetic medium containing YNB (67%) plus 2% glycerol (150 ml) and a growth at + 30 ° C was used. during 36 h. The fermentation conditions were as follows: the temperature was + 30 ° C; the pH of 5.0 was maintained using 3.5 N NH 4 OH and 2 N HCl; dissolved oxygen from 20 to 40% air saturation; propylene glycol 2000 was used as an antifoam. 20 Growth was checked at regular intervals taking OD at 600 nm. The Agoo reached a maximum of 50-60 in 24 h. At this point, the growth phase of the batch was finished according to what was indicated by the increased dissolved oxygen levels.

The growth phase was immediately followed by the induction phase. During this phase, methanol containing 12 ml / L of PTMl salts was fed. The methanol feed rate was 6 μl / h during the first 10-12 h after which it was gradually increased in increments of 6 ml / h every 7-8 h to a maximum of 36 ml / h. Ammonia was used to control the pH, which acted as a source of nitrogen. The methanol accumulation was verified every 6-8 h by adding the dissolved dissolved oxygen and it was found to be limited throughout the induction phase. The OD at 600 nm was increased from 50-60 to 150-170 during the 86 h of methanol feed. Yeast extract and peptone were added every 24 h to make a final concentration of 0.25% and 0.5% respectively. Samples were taken at 24 h intervals and verified by the enzymatic activity of BSSL in the cell-free broth. The broth was also subjected to SD-PAGE and western blot analysis. 3. 2. Analysis of the BSSL protein secreted from culture GS115 [pARC 5799] (No. 21) that grew in the fermentor The enzymatic activity of the BSSL in the cell-free broth was increased from 40-70 mg / l (native protein equivalents) in 24 h to a maximum of 200-227.0 mg / l (native protein equivalents) at the end of 86- 90 h. He * SDS-PAGE analysis of the cell-free broth showed the band stained with the band stained with prominent coomassie blue of a molecular weight of 116 kDa. The identity of the band was confirmed by the Western spotting performed as described in Section 1.7 for the native BSSL. 3. 3. Purification of secreted recombinant BSSL in the culture supernatant of clones GS115 [pARC 5799] (No. * 10 21) The P. pastoris clone GS1115 [pARC 5799] was grown and induced in the fermenter as described in Section 3.1. For the purification of recombinant BSSL, 250 ml of culture medium (induced for 90 h) were centrifuged at 12,000 x g for 30 minutes to remove all the particulate matter. The cell-free culture supernatant was ultrafiltered in an Amicon kit using a 10 kDa cut-off membrane. The salts and proteins of low Molecular weight and the peptides of the culture supernatant were removed by repeated dilution during filtration. The absorber used for such dilution was 5 mM Barbitol pH 7.4. After concentration of the culture supernatant, the retained fraction was reconstituted to 250 ml using Barbitol 5 mM, pH 7.4 and 50 mM NaCl and loaded onto a Heparin-Sepharose column (bed volume of 15 ml) What is pre-balanced with the same shock absorber. The sample was loaded at a flow rate of 10 ml / hr. After loading the column was washed with 5 mM Barbitol, pH 7. 4 and 0.1 M NaCl (200 μl wash buffer) until an absorbance at 250 nm was reached below the detection level. The BSSL was diluted 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. The fractions (2.5 ml) were collected and verified for the eluted protein by verifying the absorbance at 260 nm. The fractions containing protein were assayed for the enzymatic activity of BSSL. The appropriate fractions were analyzed on 8.0% SDS-PAGE to verify the profile of purification. 3. 4 Characterization of purified recombinant BSSL the culture supernatant of GS115 [pARC 5799] 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 approximately 116 kDa as determined by SDS-PAGE and Western blot analysis. When the samples became overloaded ^ for SDS-PAGE analysis a low molecular weight protein band could be detected by staining with Blue Brilliant of Coomassie which was not achieved with the spotted Western. The purified protein was subjected to N-terminal analysis in an automated protein sequencer. The results showed that the protein was processed appropriately from the native signal peptide and the recombinant £ * protein has the N-terminal sequence AK L G AV Y. found that the specific activity of the purified recombinant protein was similar to that of the native protein.

EXAMPLE 4: Expression of the BSSL-C in Pichia pastoris GS115 4.1. Construction of the pARC 5797 The cDNA sequence coding for the BSSL variant of BSSL-C was fused at its 5 'end with the The sequence encoding the peptide is signaled from the SUC2 gene product of S. cerevisiae (invertase), which maintains the integrity of the open reading frame that starts at the first ATG codon of the invertase signal peptide. This genetic fusion construct was initially cloned in the expression vector pSCW 231 of 5. cerevisiae (pSCW 231 is a low copy number yeast expression vector and expression is lowered control of the constitutive ADH1 promoter) between the £ coRI and BamHI site to generate the vector of expression pARC 0788. The cDNA of the fusion gene was further subcloned into the expression vector pDM 148 of P. pastoris (described in Section 1.2) by releasing the appropriate 1.8 kb fragment by digestion with EcoRI and BamHI from pARC 0788 and subcloning the fragment in pDM 148 digested with £ coRI # 10 and BamHI. The resulting construct pARC 5790 was digested with BamHI and a double-stranded oligonucleotide linker of the physical structure BamHI-.EcoRI-BaípHI was ligated to generate the ARC 5796 construct essentially to isolate the cDNA fragment from the fusion gene, following the described strategy in Section 2.1. Finally, the 1.8 kb fragment containing the * invertase signal peptide / fusion gene BSSL-C was released from pARC 5796 by digestion with EcoRI and was cloned into the pHIL D4 at the coRI site. By analyzing Appropriate restriction of the expression vector containing the insert in the appropriate orientation was identified and designated as pARC 5797 (NCIMB 40722). 4. 2 Expression of the recombinant BSSL-C of P. pastoris * To express the recombinant BSSL-C of P. pastoris, the P. pastori host GS115 was transformed with pARC 5797 by the method described in Sections 1.3 and 2.2. Transformants were verified for lipase production by the method described in Sections 1.4 and 2.2. A single transformant (No. 3) was extracted based on the high capacity of lipase production by the detection method $ 10 test on lipase plate and was further analyzed for the production of BSSL enzymatic activity in the culture supernatant following essentially the method described in Sections 1.6 and 2.3. As shown in Table 1, the culture supernatant of GS115 (pARC 5797) (No. 3) had enzymatic activity of BSSL and the amount was progressively increased up to 72 h after induction. 4. 3 SDS-PAGE and western blot analysis of the culture supernatant of transformer GS115 [pARC 5797] (No. 3) 20 The culture supernatant collected at various times as described in Section 4.2 was subjected to SDS-PAGE and analysis of western spots as described in Sections 1.7 and 2.4. From the profile of SDS-PAGE it was estimated that approximately 75-80% of the total extracellular protein was BSSL-C. The molecular weight of the protein estimated from * of the SDS-PAGE analysis was approximately 66 kDa. In the western blot analysis only two bands (doublet) were found around 66 kDa that were 5 in unreactive and in this way the expression of the recombinant BSSL-C was confirmed.

COMPARATIVE EXAMPLE: Expression of BSSL in S. cerevisiae Attempts were made to express BSSL in Saccharomyces cerevisiae. The BSSL was poorly secreted in S. Cerevisiae and the native signal peptide do not work efficiently. In addition, the native signal peptide was not cleaved from the mature protein in S. cerevisiae. 15 REFERENCES # Abouakil, N., Rogalska, E., Bonicel, J. and Lombardo, D. (1988) Biochim. Biophys. Acta. 961, 299-308. Baba, T., Downs, D., Jackson, K. W., Tang, J. and Wang, C-S (1991) Biochemistry 30, 50-510.

Bláckberg, L. and He ell, 0. (1981) Eur. J. Biochem. 116, 221-25 25.

Bláckberg, L., Angquist, K. A. and Hernell, O. (1987) FEBS Lett. 217, 37-41.

Cregg, J. M. et al. (1987) Bio / Technology 5, 479-485.

Ellis, S. B. et al. (1985) Mol. Cell. Biol. 5, 1111-1121.

Fredrikzon, B., Hernell, O., Bláckberg, L. and Olivecrona, T. (1978) Pediatric Res. 12, 1048-1052.

Hansson, L., Bláckberg, L., Edlund, M., Lundberg, L, Stromqvist, M. and Hernell, 0. (1993) J. Biol. Chem. 268, 26692-26698.

Hernell, O. and Olivecrona, T. (1974) Biochim. Biophys. Acta 369, 234-244.

Hernell, O., Bláckberg, L. and Olivecrona, T. (1989) in: Textbook of gastroenterology and nutrition in infancy (Lebenthal, E, ed.) 347-354, Raven Press, NY.

Hernell, O. and Bláckberg, L. (1982) Pediatric Res. 16, 882-885.

HUÍ, D. Y. and Kissel, J. A. (1990) FEBS Letters 276, 131-134.

Kingsman, et al. (1985) Biotechnology and Genetic Engineering Reviews 3,377-416.

Nilsson, J., Bláckberg, L., Carlsson, P., Enerbáck, S., Hernell, O. and Bjursell, G. (1990) Eur. J. Biochem. 192, 543-550.

Reue, K., Zambaux, J., Wong, H., Lee, G., Lee, TH, Ronk, M., Shively, JE, Sternby, B., Borgstrom, B., Amis, D. and Scholtz, MC (1991) J. Lipid. Res. 32, 267-276.

Wang, C-S, and Hartsuck, J. A. (1993) Biochim. Biphys Acta 1166, 1-19.

DEPOSIT OF MICROORGANISMS The following plasmids, transformed into Pichia pastoris cultures, have been deposited under the Budapest Treaty in the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, Scotland, UK. The deposit date is May 2, 1995.

TABLE 1 Enzymatic activity of the culture supernatants of Pichia pastoris transformants LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: ASTRA AB (B) STREET: Vástra Malarehamnen 9 (C) CITY: Sódertálje (E) COUNTRY: Sweden '(F) POSTAL CODE (ZIP): S- 151 85 (G) TELEPHONE: + 46-8-553 260 00 (H) TELEFAX: + 46-8-553 288 20 (I) TELEX: 19237 astra s (ii) TITLE OF THE INVENTION: DNA Molecules for the Expression of Polypeptides (iii) SEQUENCE NUMBER: 4 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM PC compatible (C) OPERATIONAL SITEMA: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2428 base pairs 5 (B) TYPE: nucleic acid (C) HEBRA: double CD) TOPOLOGY : linear (ii) TYPE OF MOLECULE: cDNA to mRNA 10 (iii) HYPOTHETIC: NO (iv) ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (F) TYPE OF TISSUE: mammary gland (ix) CHARACTERISTICS: 20 (A) NAME / KEY: CDS (B) LOCATION: 82..2319 (D) OTHER INFORMATION: / product = "lipase stimulated by bile salts" (ix) CHARACTERISTICS: '(A) NAME / KEY: exon (B) LOCATION: 985..1173 (ix) CHARACTERISTICS: (A) NAME / KEY: exon (B) LOCATION: 1174..1377 (ix) CHARACTERISTICS: 10 (A) NAME / KEY: exon (B) LOCATION: 1378..1575 (ix) CHARACTERISTICS: (A) NAME / KEY: exon 15 (B) LOCATION: 1576..2415 (ix) FEATURE: (A) NAME / KEY: mat_peptide (B) LOCATION: 151..2316 [ix) CHARACTERISTICS: (A) NAME / KEY: poliA_sígnal (B) LOCATION: 2397..2402 (ix) FEATURE: $ (A) NAME / KEY: repit_region (B) LOCATION: 1756..2263 (ix) FEATURE: (A) NAME / KEY: 5 'UTR (B) LOCATION:! .. 81 (ix) FEATURE: 10 (A) NAME / KEY: repeat unit (B) LOCATION: 1756..1788 (ix) FEATURE: (A) NAME / KEY: repeat_unit 15 (B) LOCATION: 1789..1821 (ix) FEATURE: (A) NAME / KEY: repeat_unit (B) LOCATION: 1822..1854 20 (ix) CHARACTERISTICS: (A) NAME / KEY: repeat_unit (B) LOCATION: 1855..1887 ^ (ix) FEATURE: 9 (A) NAME / KEY: repeat_unit (B) LOCATION: 1888..1920 (ix) CHARACTERISTICS: 5 (A) NAME / KEY: repeat_unit (B) LOCATION: 1921..1953 (ix) FEATURE: £ ffc (A) NAME / KEY: repeat_unit (B) LOCATION: 1954. 1986 (ix) CHARACTERISTICS: (A) NAME / KEY: repeat_unit (B) LOCATION: 1987. .2019 15 (ix) CHARACTERISTICS: - (A) NAME / KEY: repeat_unit (B) LOCATION: 2020..2052 (ix) FEATURE: (A) NAME / KEY: repeat_unit (B) LOCATION: 2053..2085 (ix) FEATURE: 25 (A) NAME / KEY: repeat unit (B) LOCATION.-2086..2118 (ix) FEATURE: (A) NAME / KEY: repeat_unit 5 (B) LOCATION: 2119..2151 (ix) FEATURE: (A) NAME / KEY: repeat_unit (B) LOCATION: 2152..2184 • 10 (ix) FEATURE: (A) NAME / KEY: repeat_unit (B) LOCATION: 2185..2217 (ix) FEATURE: (A) NAME / KEY: repeat_unit (B) LOCATION: 2218..2250 (ix) FEATURE: 20 (A) NAME / KEY: repeat_unit (B) LOCATION: 2251..2283 (x) PUBLICATION INFORMATION: (A) AUTHORS: Nilsson, Jeanette 25 Bláckberg, Lars Carlsson, Peter Enerbáck, Sven Hernell, Olle Bjursell, Gunnar B) TITLE: Cloning of lipase cDNA stimulated by bile salts of human milk and evidence of its identity with carboxylic ester hydrolase. 10 (C) JOURNAL Eur. J. Biochem. (D) VOLUME 192 (F) PAGES 543-550 (G) DATE: September-1990 xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ? ccTTCTOT? TC? OTT ?? crr CTCA ß? oc ?? 8 ?? c? oc? GTc 2? T ??? GC ??? ß 60? CTTT? TTC? TCC? ß? OGCT O? T? CTC? CC? TO OOO ccc: C ?? CTß ßTT 111 Mae au Thr Mat Oly? g - «-. Without Uu Val -23 -20 -15 GTG TTG OGC CTC? CC TOC TGC TOO GC? GT8 GCO? GT zz sß ?? ß ero 159 Val Lau Gly Lau Thr Cys Cys Trp? Val? Sar Alt,? Lyt Lau -10 -5 1 20 OGC GCC GTG T? C? C? C ?? GGT GGG TTC GTß G ?? GGC m ?? T ?? s? ß 207 Gly? The Val Tyr Thr Glu Gly Gly Pha Val Glu Gly Vil? An Lys Lys 5 10 15 CTC OGC CTC CTO OOT G? C TCT OTO G? C? TC TTC ?? G; z? TC ccc TTC 255 Lau Oly Lau Lau Gly? Ap Sar Val? Ap Xla Pha Lys _y? L? Pro Ph? 20 25 30 35 GC? GCT CCC? CC ?? ß GCC CTG G ?? ?? T CCT C? G CC? r CCT ßßc toß 303? the? Pro Thr Lys? the Lau Glu? an Pro Ola Pro:? Pro ßly Trp 40 45 50 C ?? OQO? CC CTO ?? ß CCC ?? ß ?? C TTC ?? ß ?? ß? C? T5 CTß CAS CCC 351 Cln Gly Thr Lau Lya? Lya? Aa Pha Ly? Lya? Rg Cy? Lau Wave? 25 55 «0 CS? CC? TC? CC C? ß G? C? CC? CC T? C 000 G? T G ?? G? C T5C CT? T? C CTC 399 '(&Thr? Thr Gln? ßp Sar Thr Tyr Gly? ßp Clu? ap Cyß Lau Tyr Lau 70 75 td? C? TT TGG GTß CCC C? ß OGC? ßß? GC? GTC TCC CSC CAC CTß CCC 447? An Xla Trp Val Pro Oln Gly? Rg Lya Oln Val Sar? Rg? Ap Lau Pro 15 90 95 GTT? TO? TC TOO? TC T? T OCA OGC CCC TTC CTC? TG C9? TCC OOC C? T 495 Val Mat Xla Trp Xla Tyr ßly Cly? The Pha Lau Mßt Gly Sar ßly Bia 100 IOS 110 115 GOG GCC ?? C TTC CTC ?? C ?? CT? C CTG T? TG? C GGC G? OG? C? TC GCC 543 Gly? La? ßn Pha Lau? ßn? ßn Tyr Lau Tyr? ßp Gly Glu Glu? La? The 120 12S 130? C? CGC GG? ? C CTC? TC GTG GTC? CC TTC? C T? C CGT GTC CGC CCC 591 Thr? Rg Gly? An Val lia Val Val Thir Pha? ßn Tyr? Rg Val Gly Pro 135 140 145 CTT GGG TTC CTC? GC? CT GGG G? C GCC ?? T CTG CC? GGT ?? CT? T GGC 639 Lau Gly Pha Lau Sar Thr Gly? Sp? The? ßn Lau Pro Gly? Sn Tyr Oly 150 1SS 160 10 CTT CGG C? TC? GC? C? TG GCC? TT GCT TGG GTG? ? G? GO ?? T? TC CCG 687 Lau? Rg? ßp ßln His Mat? The Xla? The Trp Val Lys? Rg? Sn? La? The 165 170 175 GCC TTC GGG GGG G? C CCC ?? C? ? C? TC? CO CTC TTC GGC G? G TCT GCT 73S? Pha Gly Sly? ßp Pro? ßn? Sn XI * Thr Lau Pha Gly Glu Sar? 180 185 190 195 GCA GCT CCC ACC GTC TCT CTO ACC ACC CTC TCC CCC T? C ?? C ?? C OGC 783 Gly Cly? La Sar Val S * r Lau Cln Thr Lau Ser Pro Tyr? ßn Ly? Oly 200 205 210 CTC? TC CGG CG? CCC? TC? GC C? C? GC GGC CTG GCC CTO? GT CCC TOO 831 Lau Xla? Rg? Rg? The Xla Sar Cln S * r Oly Val? La Lau S * r Pro Trp 15 215 220 225 GTC? TC CAC A ?? ?? C CC? CTC TTC TOO CCC ??? C GTO CCT GAO ?? ß 879 Val II * Cln Lys? An Pro Lau Ph? Trp? Lys Lys Val? Glu Ly? 230 23S 240 GTG CGT TGC CCT GTG GGT G? T CCC GCC? GG? TG GCC CAG TGT CTß ?? ß 927 Val Cly Cys Pro Val Cly? ßp? La? La? Rg Mat? Cln Cy? Lau Lys 245 250 255 GTT? CT G? T CCC CG? GCC CTO? CG CTO GCC T? T ?? O OTO CCG CTß GCA 975 Val Thr? ßp Pro? Rg? The Lau Thr Leu? The Tyr Ly? Val Pro Lau? The 260 265 270 275 20 GGC CTG G? GT? C CCC? TG CTO C? CT? T CTO OGC TTC GTC CCT GTC? TT 1023 Gly Lau Glu Tyr Pro Mßt Lau Ris Tyr Val Oly Phß Val Pro Val? La 280 28S 290 GAT GGA GAC TTC ATC CCC GCT GAC CCO? TC ?? C CTO T? C GCC AAC GCC 1071? ßp Gly? ßp Phß Xla Pro The? ßp Pro? the Aßn Lau Tyr Ala? ßn? the 295 300 305 GCC G? C? TC C? CT? T? T? OC? GCC? CC ?? C ?? C? T? G? C GGC C? C? TC 1119? La? ßp Xla? ßp Tyr? The? The Gly Thr? An? ßn Mat? Ap Gly Hi? Lia 310 315 320 TTC OCC ? GC? TC G? C? T? CCT CCC? TC ?? C ?? G GGC A? C ?? C ??? CTC 1167 Ph *? La Sar? La? Ap Mat Pro? La? An Lya ßly? An Lyß Lyß Val 32S 330 - 335 1215 OOO CTC? G? OGC GCC ?? ß? CO? CC TTT O? T GTC TAC ACC CAO TCC TOO 1263 Oly Lau? Rg ßly? Lyß Thr Thr Pha? ßp Val Tyr Thr ßlu S * r Trp 360 365 370 OCC C? ß C? C CC? TCC C? CC? C ?? T ?? C? AO ?? C? CT OTO CT? CAC TTT 1311 Wing Wave? Pro Pro Wave Olu? A Lys Ly? Lya Thr Val Val? Ap Pha 375 380 385 G? O ACC C? T GTC CTC TTC CTC CTG CCC? CC G? C? TT GCC CT? GCC C? C 1359 Glu Thr? Sp Val Lau Pha Lau Val Pro Thr Glu Has Lau la Gln 390 395 400 CAC ACÁ GCC AAT GCC AAG ACT GCC AAO ACC TAC CCC TAC CTG TTT TCC 1407 Hiß Arg. Ala Aßn Ala Lys S * r? The Lys Thr Tyr Wing T r Lau Pha Sar 405 410 415 CAT CCC TCT CGG ATG CCC GTC TAC CCC ??? TGG GTG CGG CCC C? C CAT 1455 His Pro Sar Arg Mac Pro Val Tyr Pro Lys Trp Val Gly? La? ßp Hiß 420 425 430 435 10 CC? C? TG? C? TT C? CT? C CTT TTC OGC '?? ß CCC TTC GCC? CC CCC? CG 1503 Wing? Sp? ßp Xla Gln Tyr Val Pha Cly Lys Pro Ph *? Thr Pro Thr 440 445 450 CCC TAC CCC CCC CA? C? C? GC? C? GTC TCT ?? OCC? TC? TC CCC T? C 1551 Cly Tyr? Rg Pro Cln? ßp? Rg Thr Val Sar Lya? Mat? La? Tyr 455 460 465 TGG ACC A? C TTT CCC ??? ? C? CGC G? C CCC? C? TG GGC C? C TCC GCT 1599 Trp Thr? ßn Ph *? L * Lyß Thr Cly? ßp Pro? ßn Mat Cly? ßp Sar? La 470 475 480 CTC CCC ACÁ CAC TGG GA ? CCC T? C? CT ACG G ?? ?? C? GC GCC T? C CTC 1647, - Val Pro Thr Ki? Trp Clu Pro Tyr Thr Thr Clu? ßn Sar Oly Tyr Lau - 485 490 49S G? C? TC? CC AAC A? O? T? GGC? GC ACC TCC? Tß ?? ß CCC ACC CTß? G? 1695 Glu II * Thr Lyß Lyß Mßt Oly S * r S * r Sar Mßt Lys? Rg S * r Lau? Rg ^^ 8 * 500 505 510 SIS? Cc? C rrc ero ecc TAC TOO? Cc ere ACC TAT CTG cea ero ecc ACÁ 1743 Thr Aan Pha Lau? rg Tyr Trp Thr Lau Thr Tyr Lau? la Lau Pro Thr 520 525 530 OTO? CC G? C CAC CAO CCC? CC CCT CTß CCC CCC? C? OCC C? C TCC GAO 1791 Val Thr? Ap ßln ßlu? The Thr Pro Val Pro Pro ttox ßly? ßp Sar ßlu 535 540 545 20 GCC ACT CCC OTO CCC CCC ACO GGT O? C TCC CAO? CC GCC CCC OTO CCO 1839 ? Thr Pro Val Pro Pro Thr Oly? sp Sar ßlu Thx? Pro Val Pro 550 555 560 CCC ACÓ GßT ß? C TCC GCG CCC CCC CCC CTß CCO CCC? CO OCT GAC TCC 1887 Pro Thr Oly? ap Sar Oly? Pro Pro Val Pro Pro Thr ßly? ap Sar 565 570 S7S OCC GCC CCC CCC GTG CCG CCC? CG OCT GAC TCC OOO CCC CCC CCC GTß 1935 Cly? the Pro Pro Val Pro Pro Thr Gly? ßp S «r Cly? Pro Pro Val 580 58S 590 595 CCC CCC ACC GCT G? C TCC GOC GCC CCC CCC GTG CCG CCC? CO OGT GAC 1983 Pro Pro Thr Gly Aap Sar Cly? Pro Pro Val Pro Pro Thr ?ly? 600 600 60S CCC CCC 2031 Pro Pro ACO OOT 2079 Thr Oly C? C CCC COC CCC CCC CCC CTO CCC CCC ACO OOT G? C TCC COC eCC CCC 2127? Ap? The Oly Pro Pro Pro Val Pro Pro Thr Oly? Ap M ?ly? The Pro «45 C50« 55 CCC GTO CCO CCC ACO GGT GAC TCC GCC GCC CCC CCC CTC ACC CCC ACC 2175 Pro Val Pro Pro Thr Cly? Ap S * r Cly? Pro Pro Val Thr Pro Thr 660 665 670 675 GCT G? C TCC CAG ACC GCC CCC GTG CCG CCC? CG GCT C? C TCC GCG GCC 2223 Gly Asp Sar Clu Thr? Pro Val Pro Pro Thr Gly? Ap S * r Gly? 680 685 «} • } CCC CCC 2271 Pro Pro TTT TAC 2319 Pr.a * CCTCCCATC? GCCTTOGT? T C ?? C? 8CC? C? C? GTGCG? CCCC? CCGG CTCCCC "XC 2379? TCTTCAGCT CTTCCTG ?? T ??? CCCTC? T? CCCCT ???? ????????? 2 28 (2) INFORMATION FOR SEQ ID NO: 2: 15 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 746 amino acids (B) TYPE; amino acid (D) TOPOLOGY: linear 20 (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Thr Cyß 7_r Clu Gly Gly Pha Val Glu Gly Val? ßn Lyß Lyß Uu ßly Uu Uu 31 / Aßp 10 15 20 25 S * r Val? Sp lia Pha Lyß ßly II * Pro Ph *? The? Pro Thr Ly?? 30 35 Lau ßlu? An Pro Wave Pro Bia Pro ßly Trp ßln ßly Thr Uu Lys? 4S 50 55 Lya? An Pha Lya Lya? Rg Cys Lau ßln? Thr? Thr Clr. ? sp s * r 60 65 70 Thr Tyr Cly? ßp Glu? ßp Cyß Lau Tyr Uu? sn? the Trp Val? ro Cia 75 80 85 Oly? rg Ly? Gla Val Sar? rg? ap Lau Pro Val Mat? l * Trp? to Tyr 90-95 1 10000 105 »< 31y Oly? La Pha Lau Mat Cly Sar Gly Bis Gly? La? An Ph * Lau? Aa 1 0 110 115 120 Aan Tyr Uu iyr? Sp Oly Oiu ßlu lß? The Thr? Rg Oly? ßn Val l? 125 130 13S Val Val Thr Ph?? Aa Tyr? Rg Val Cly Pro Uu Oly Pha Uu Sar Thr 140 14S 150 Oly? Ap? La? Aa u Pro ßly? Aa Tyr ßly Uu? Rg? Ap ßla Bia Mat 155 160 16S? The II *? The Trp Val Lys? Rg? An lia? La? The Pha ßly ßly? Ap Pro 170 175 180 185 1 5 Aßn Aan XI * Thr Uu Ph * ßly Glu Ser Ala ßly Gly? The S «r Val Sar 190 195 200 Lau Cln Thr Uu Sar Pro Tyr? Sn Lys Gly Uu? La? Rg? Rg? La II * 205 210 215 S * r Cln S * r ßly Val? Uu S * r Pro Trp Val Xla ßln Lys? An Pro 220 225 230 Uu Phe Trp? Lya Lyß Val? ßlu Lyß Val Gly Cys Pro Val Cly 23S 240 24S? ßp? la? la? rg Mat? the Gln Cys Uu Lys Val Thr? ap Pro? rg? la 250 255 260 265 UM Thr Lau? L * Tyr Lys Val Pro Lau? The Oly Uu Glu Tyr Pro Mat 270 275 280 Lau Hi? Tyr Val Gly Pha Val Pro Val X? Ap Cly? Ap Ph * XI * Pro 285 290 295 Pro? Xle? ßn Uu Tyr? the? an? la? the? ap XI *? ap Tyr lie 300 305 310? the Cly Thr? ßn? ßn Mat? sp Gly Hiß? the Pha? the Sar lie? sp Met 315 320 325 25 Pro? la? ßn Lyß Gly? ßn Lyß Lyß Val Thr ßlu ßlu? ap Phe Tyr 330 335 340 345 Lys Leu Val Ser Glu Phe Thr II * Thr Lyß Gly Uu? rg Cly? la Lyß 350 355 360 Thr Thr Pha? Ap Val Tyr Thr Glu Sar Trp? Gln? Ap Pro Ser Gla 36S 370 375 Clu? ßn Ly? Lys Lys Thr Val Val? Ap Phe Glu Thr? Ap Val Uu Pha 380 385 390 Lau Val Pro Thr Glu Xla ? Uu? la Gla His? rg Ala? sa? the Lyß 395 400 405 Ser? the Lys Thr Tyr? the Tt Uu Phe Ser Hi? Pro Sar? rg Mat Pro 410 415 420 425 Val Tyr Pro Lys Trp Val Cly? La? Sp His? La? Ap? Sp? The Cia Tyr 430 43S 440 Val Phe Gly Lyß Pro Phe? The Thr Pro Thr Oly Tyr? Rg Pro Oln? Ap 445 4S0 4SS? Rg Thr Val Ser Lya? The Mat Xle? The Tyr Trp Thr? An Pha? The Lys 460 46S 470 Thr Gly? Ap Pro? Sa Mat ßly? Ap S * r? Val Pro Thr Hi? Trp ßlu 47S 480 4SS Pro Tyr Thr Thr ßlu? An Sar ßly Tyr u ßlu? The Thr Lya Lys Mat 490 49S 500 S05 Oly Ser Ser Met Met Lya? Rg Ser Uu? Rg Thr? An Pha Uu? Rg Tyr 510 515 S20 Trp Thr Uu Tnr Tyr Lau? The Uu Pro? R Val Thr? Ap Ola Olu? The S2S S30 S35 Thr Pro Val Pro Pro Thr Oly? ßp Ser Olu? The Thr Pro Val Pro Pro 540 545 550 Thr Gly Aßp S * r Glu Thr Ala Pro Val Pro Pro Thr Cly? Sp Ser Gly 555 560 565? Pro Pro Val Pro Pro Thr Gly? Ap Sar Cly? Pro Pro Val Pro 570 575 580 585 Pro Thr Gly? ßp S * r Gly? Pro Pro Val Pro Pro Thr Gly? Ap Ser 590 595 600 Gly? The Pro Pro Val Pro Pro Thr Gly? Ap Ser Cly? The Pro Pro Val 605 CIO 615 Pro Pro Thr Cly? Ap Ser Cly? The Pro Pro Val Pro Pro Thr Cly? Ap 620 625 630 Ser. Gly? Pro Pm Val Pro Pro Thr Gly? ßp? The ßly Pro Pro Pro 635 640 645 Val Pro Pro Thr Gly? Ap Ser ßly? The Pro Pro Val Pro Pro Thr ßly 650 655 660 665? Ap Ser Gly Pro Pro Wing Val Thr Pro Thr Gly? Ap Ser Glu Thr? La 670 675 680 Pro Val Pro Pro Thr Oly? Ap Ser Gly? Pro Pro Val Pro Pro Thr 685 690 695 Gly? Ap Ser Glu? Pro? Val Pro Pro Thr? ßp? Ap Ser Lys Glu 700 705 710? The Gln Mat Pro? The Val Xle? Rg Phe • 715 720 (2) INFORMATION FOR SEQ ID NO: 3 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 722 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear Ai) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (F) TYPE OF TISSUE: Mammary gland (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 ? Lys Lau Oly? Val Tyr Thr Olu ßly cly Ph * Val Clu ßly Val 5 10 15? an Lys Lys Lau Gly Lau Lau? ap Sar Val? ap lia Pha Lys ßly 20 25 30 Xla Pro Pba? la? the Pro Thr Lya? the Lau ßlu? aa Pro Ola Pro Ria 35 40 45 Pto ßly Trp Ola Oly Thr Lau Lys? the Lya? aa Pha Lys? rg Cy? SO SS 60 Lu ßln? the Thr Xle Thr ßln? ap S_r Thr Tyr ßly? Ap ßlu? Ap Cyß 65 70 75 80 Leu Tyr Leu? An Xla Trp Val Pro Cln Gly? Rg Lya Oln Val Ser? Rg 85 90 95 Asp Leu Pro Val Met He Trp He Tyr Gly Gly? The Ph * Uu Met Gly 100 105 110 S * r Cly Hiß Gly? the? ßn Phe Uu? ßn? ßn Tyr Uu Tyr? ßp ßly Clu 115 120 12S Clu II *? the Thr? rg Cly? an Val He Val Val Thr Pha? ßn Tyr? rg 130 135 140 Val Gly Pro Uu Gly Phe Leu Ser Thr Gly? Ap? La? An Uu Pro Cly 145 150 155 160? Sn Tyr Gly Uu? Rg? Ap Gln H? Met Met? The? Trp Val Ly? Rg 165 170 175? Sn He? La? the Pha Gly Gly? ßp Pro? ßn? ßn? the Thr Uu Phe Cly 180 185 190 Glu Ser? the Cly Cly? the Ser Val Ser Uu Gln Thr Uu Ser Pro Tyr 195 200 205? ßn Lyß Cly Uu He? rg? rg? the Xle S * r Gln Ser Gly Val? the Uu 210 215 220 Ser Pro Trp Val Xle Gln Lya? ßn Pro Uu Phe Trp? the Ly? Ly? Val 225 230 235 240? the Glu Ly? Val Gly Cy? Pro Val Cly? ßp? la? la? rg Met? Cln 245 250 255 Cys Leu Lya Val Thr? ßp Pro? Rg? The Uu Thr Uu? L * Tyr Lyß Val 260 265 270 Pro Leu? The Gly Lau Glu Tyr Pro Met Uu Hi? Tyr Val Gly Phe Val 275 280 28? Pro Val He? ßp Gly ? ßp Phe II * Pro? la? ßp Pro XI *? ßn Uu Tyr 290 295 300? the? ßn? la? ßp Xle? ap Tyr Xle? the Cly Thr? aa? aa Met? ßp 305 310 315 320 Cly Kiß He Phe? The Ser? Le? ßp Met Pro? La Ha? An Lya Oly? ßn 325 330 335 Lyß Lyß Val Thr Clu Clu? ßp Pha Tyr Lyß Uu Val Sar Clu Phe Thr 340 345 350 le Thr Lyß Oly Uu? Rg Oly? The Lyß Thr Thr Pha? Ap Val Tyr Thr 355 360 3CS Glu Ser Trp? The Cln? Ap Pro S * r Oln Glu? An Lys Lys Lyß Thr Val 370 375 380 Val? Ap Phe Clu Thr? Ep Val Leu Phe Uu Val Pro Thr Glu? Le? The 385 390 395 400 Uu? The Gln Bia? Rg? The? An? The Lya Ser? The Lya Thr Tyr? The Tyr 405 410 415 Uu h * Sar Bis Pro s * r? Rg Mßt Pro Val Tyr Pro Lya Trp Val Oly 420 425 430,? The? ßp Hiß? The? Ap? Ap Xle Gln Tyr Val Ph * Gly Ly? Pro Ph *? La 435 440 445 Thr Pro Thr Gly Tyr? Rg Pro Gln? ßp? Rg Thr Val Ser Lyß? The Mßt 450 4S5 460 He? Tyr Trp Thr? ßn Phß? L * Lys Thr Cly? Sp Pro? ßn Met Cly .65 470 475 480? ßp S «r? Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu? ßn Ser 48S 490 495 Tyr Uu Glu He Thr Lyß Lyß Met Gly Ser Ser Ser Met Lyß? Rg 500 505 510 S «r L« u? Rg Thr? ßn Phe Leu? Rg Tyr Trp Thr Leu Thr Tyr Uu? La 515 520 525 - * u Prc Thr Val Thr? ßp Gln Glu? The Thr Pro \ 'al Pro Pro Thr Gly SK 535 540? Ap S «r Glu Ala Thr Pro Val Pro Pro Thr Gly? Ap Ser Clu Thr? La 545 550 555 560 ? Pro Val Pro Pro Thr Gly? ap Ser Gly? the Pro Pro Val Pro Pro Thr 565 570 575? s Sar Gly? the Pro Pro Pro Pro. Thr Cly? ßp Ser Cly? the 580 585 590 rro Pro Val Pro Pro Thr Gly? ßp Ser Gly? The Pro Pro Val Pro Pro 595 600 605 Gly? ßp Sar Gly? The Pro Pro Val Pro Pro Thr Gly? ßp Ser Cly 610 61S 620? Pro Pro Val Pro Pro Thr Cly? ßp Ser Gly? Pro Pro Val Pro 1.5 630 635 640 Pro Thr Gly? ßp? The Gly Pro Pro Pro Val Pro Pro Thr Cly? ßp Ser 64S 650 655 31y? L * Pro Pro Val Pro Pro Thr Gly Aßp S * r Gly Ala Pro Pro Val 660 665 670 7 > _r Pro Thr Gly Aßp Ser Glu Thr? Pro Val Pro Pro Thr Gly? ap 675 680 685 S * r Gly Pro Pro Val Pro Pro Thr Gly? ßp Ser Glu? la? Pro 690 695 700 Val Pro Pro Thr ? ap? ap Ser Lyß Glu? the Gln Mat Pro? the Val He 705 710 715 720? Rg Pha INFORMATION FOR SEQ ID NO: 4: SEQUENCE ARCHTERISTICS: (A) LENGTH: 568 amino acids (B) TYPE: amino acid (C) HEBRA: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (F) TYPE OF TISSUE: Mammary gland (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCALI ZATION: 1..568 (D) OTHER INFORMATION: / level = Variant_C (x) PUBLICATION INFORMATION: (A) AUTHORS: Hansson, Lennart Blackberg, Lars Edlund, Michael Lundberg, Lennart Stromqvist, Mats Hernell, Olle (B) TITLE: Lipase stimulated by bile salts of recombinant human milk. (C) REVIEW: J. Biol. Chem. (D) VOLUME: 268 (F) PAGES: 26692-26698 (G) DATE: December 15 - 1993 ! xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 At * Lyß Uu Gly? Val Tyr Thr Glu Cly Gly Ph? Val Glu Oly Val 1 5 10 15? ßn Lyß Lyß Uu Gly Uu Leu Gly? Sp Ser Val? Ap Xla Pha Lyß ßly 20 25 30 Xle Pro Phe? La? the Pro Thr Lyß? the Lau ßlu? an Pro ßln Pro Hiß 35 40 45 Pro Gly Trp Gln Cly Thr Leu Lyß? the Lya? ßn Phe Lys Lya? rg Cyß 50 55 60 Uu Gln? the Thr Xla Thr Gln? ap * x Thr Tyr Oly? ßp Olu? ßp Cyß 65 70 75 80 Uu Tyr Uu? ßn X the Trp Val Pro Oln Gly? Rg Lya Gln Val Sar? Rg 85 90 95? ßp Uu Pro Val Mat Xle Trp Xle Tyr Gly Gly? L * Pha Uu Met Gly 100 105 110 Ser Gly Hiß Gly? the? an Pha Uu? an? sn Tyr Uu Tyr? ap Oly Clu 115 120 12S Clu He? the Thr? rg Oly? an Val Xle Val Val Thr Pha? aa Tyr? rg 130 135 140 Val oly Pro Uu Oly Ph * Uu Sar Thr Oly? Ap? La? An Uu Pro ßly 145 150 155 160? An Tyr Oly Uu? Rg? Ap Gla Bia Mat? La? La? L * Trp Val Lya? Rg 16S 170 175? An Ha? La? the Pha Oly Gly? sp Pro? sn? ßa Xla Thr Uu Pha Oly 180 185 190 Olu Ser? the Oly Cly? the Ser Val Ser Uu Wave Thr Lau S? r Pro Tyr 195 200 205? ßn Lyß Oly Uu XI *? rg? rg? l * lie Ser Oln Ser Gly Val? la Uu 210 215 220 Ser Pro Trp Val He Gln Lyß? ßn Pro L «u Ph? Trp? Lya Ly? Val 225 230 235 240? Glu Ly? Val Gly Cy? Pro Val Gly? Sp? La? La? Rg Met? Cln 245 2S0 25S Lyß Val Pro Uu Wing Gly Leu Glu Tyr Pro Met Lau Hiß Tyr Val Cly Pha Val 275 280 28S Pro Val He? ßp Gly? ßp Pha He Pro? La? ßp Pro Ha? Sn Uu Tyr 290 295 300? La? ßn? la? la? ap He? ap Tyr ll *? the Gly Thr? an? ßn Mat? ap 305 310 315 320 Cly Hit He Ph *? the Being He? ßp Met Pro? the He? ßa Lyß Cly? an 32S 330 335 Lya Lys Val Thr Clu Clu? ßp Pha Tyr Lyß Uu Val Ser Clu Phe Thr 340 345 350 He Thr Lyß Gly Leu? Rg Gly? The Lyß Thr Thr Ph * Aßp Val Tyr Thr 355 360 • 365 Glu S «r Trp Ala Gln? ßp Pro S * r Gln Glu? ßn Lys Lyß Lyß Thr Val * 370 375 380 10 Val? ßp Pha Glu Thr? ßp Val Uu Pha L «Val Pro Thr Clu Ha? 385 390 395 400 Uu? Gln Hiß? Rg? La? ßn? L * Lyß Sar? Lyß Thr Tyr ? the Tyr 405 410 415 Lau Pha Sar Kia Pro Sar? rg Met Pro Val Tyr Pro Lya Trp Val ßly 420 425 430? the? ßp Hiß? the? sp? ßp 11 * ßln Tyr Val Phe ßly Lyß Pro Phe? the 435 440 445 Thr Pro Thr ßly Tyr? Rg Pro ßln? Ap? Rg Thr Val Ser Lya? La Mat 450 455 460 He? The Tyr Trp Thr? ßn Pha? The Lys Thr ßly? ßp Pro? ßn Mat ßly 465 470 475 480? Ap Sar? The Val Pro Thr Ria Trp Glu Pro Tyr Thr Thr Glu? Aa Ser 485 490 49S Gly Tyr Uu ßlu He Thr Lya Lya Met Cly Ser Ser Met Lys? Rg 500 505 510 Ser Uu? Rg Thr? Aa Pha Uu? Rg Tyr Trp Thr Uu Thr Tyr Uu? La 515 520 525 20 Uu Pro Thr Val Thr ? app Ola ßly? the Pro Pro Val Pro Pro Thr ßly 530 S3S 540? ap St ßly? the Pro Pro Val Pro Pro Thr ßly? m ^ Ser Lya ßlu? the 545 550 5SS SCO ßla Mat Pro? the Val Xla? rg Pha 5C5 It is noted that with reference to this date, the # best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following: *

Claims (14)

1. A DNA molecule, characterized in that it comprises: (a) a region encoding a polypeptide which is the human BSSL or a biologically active variant thereof; (b) attached to the 5 'end of the region encoding the polypeptide, a region encoding a signal peptide capable of directing the secretion of the polypeptide from Pi chia pastoris cells transformed with the DNA molecule; and (c) operably linked to the coding regions defined in (a) and (b), the methanol oxidase promoter of Pichia pastori s or a functionally equivalent promoter.

2. The DNA molecule according to claim 1, characterized in that the signal peptide 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 List of Sequences.

3. The DNA molecule according to claim 1, characterized in that the signal peptide comprises an invertase signal peptide of Saccharomyces cerevisiae.

4. The DNA molecule according to any of claims 1 to 3, characterized in that it codes for a biologically active variant of the human BSSL, in which at least one of the repeated units of 11 amino acids, the repeated units are indicated in the SEQ ID NO: 1, is deleted. *

5. The DNA molecule according to any of claims 1 to 4, characterized in that it encodes a polypeptide which has BSSL activity and an amino acid sequence which is at least 15 95% homologous to the sequence according to SEQ ID NO: 3 or SEQ ID NO: 4.

6. The DNA molecule according to any of claims 1 to 5, characterized 20 because it encodes a polypeptide which has the amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4.

7. A vector, characterized in that it comprises a # DNA molecule according to any of claims 1 to 6.

8. A duplicatable expression vector, according to claim 7, characterized in that it is capable of mediating the expression of human BSSL or a biologically active variant thereof, in Pichia pastoris cells. *

9 The vector according to claim 8, characterized in that it is the plasmid vector pARC 5771 (NCIMB 40721), pARC 5799 (NCIMB 40723) or pARC 5797 (NCIMB 40722).

10. Host cells of the genus Pichia, characterized in that they are transformed with a vector according to any of claims 7 to 9.

11. The host cells according to claim 10, characterized in that they are Pichia pastoris cells.

12. The host cells in accordance with * claim 11, characterized because they are Pichia pastoris cells of strain GS115.

13. The host cells according to claim 12, characterized in that they are PPF-1 [pARC 5771] (NCIMB 40721), GS115 [pARC 5799] (NCIMB 40723) or GS115 [pARC 5797 (NCIMB 40722). • 10

14. A process for the production of a polypeptide which is human BSSL, or a biologically active variant thereof, characterized in that it comprises culturing host cells according to any of claims 10 to 13 under conditions 15 by which the polypeptide is secreted into the culture medium, and recovering the polypeptide from the culture medium.