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  • C12N15/09—Recombinant DNA-technology
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

• BSSL bile salt-stimulated Lipase
• 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.
• BSSL bile salt-stimulated lipase
• Bile salt-stimulated lipase (BSSL; EC 3.1.1.1) (for a review see Wang & 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).
• 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 (Blackberg et al., 1987). It accounts for approximately 1% of the total milk protein (Blackberg & Hernell, 1981).
• 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 (US 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.
• 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 US 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.
• 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).
• 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 lines.
• a suitable host such as E. coli, Saccharomyces cerevisiae, or mammalian cell lines.
• heterologous expression systems could be envisaged.
• 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.
• 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.
• 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.
• 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).
• Sacharomyces cerevisiae as a host is that the recombinant proteins tend to be overglycosylated which could affect activity of glycosylated mammalian proteins.
• 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). Use of Pichia pastoris is known in the art as a host for the expression of e.g.
• 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).
• 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.
• 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.
• human BSSL and the variant BSSL-C 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 translocate the mature protein into the culture medium as an active, properly processed form. DESCRIP ⁇ ON OF THE INVENTION
• the invention provides a DNA molecule comprising:
• 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.
• 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-spe ⁇ f ⁇ c 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).
• 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 nucleotide sequence indicated as a "repeat unit” under the heading “(ix) FEATURE” in "INFORMATION FOR SEQ ID NO: 1" in the Sequence Listing.
• 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).
• 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 907c homologous, preferably at least 95% homologous, with the amino acid sequence shown as SEQ ID NO: 3 or 4 in the Sequence Listing.
• 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.
• it can be a peptide which comprises a Saccharomyces cerevisiae invertase signal peptide.
• the invention provides a vector comprising a DNA molecule as defined above.
• 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.
• a vector can e.g. be the plasmid vector pARC 5771 (NCTMB 40721), pARC 5799 (NCIMB 40723) or pARC 5797 (NCIMB 40722).
• the invention provides a host cell culture comprising cells of the genus Pichia transformed with a DNA molecule or a vector as defined above.
• 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).
• 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.
• 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- Sacl fragment.
• pTZ19R Pharmacia
• the cloning of NSP-BSSL cDNA into S. cerevisiae expression vector pSCW 231 obtained from professor L. Prakash, University of Rochester, NY, 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.
• NSP-BSSL cDNA was cloned into pYES 2.0 (Invitrogen, USA) as an EcoW-Sphl fragment from pTZ19R-SP-BSSL.
• the excess 89 base pairs between the EcoRI and Ncol 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 Ncol site which was immediately followed by the regenerated EcoRI site in frame with the remaining NSP-BSSL sequence.
• 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 (MOXl) gene were isolated by PCR and placed in tandem in the multiple cloning sequence (MCS) of E. coli vector pSK + (available from Stratagene, USA).
• 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.
• the expression cassette can be cleaved from the rest of the pSK ⁇ vector by digestion with Notl restriction enzyme.
• the 5'-UTR of MOXl of P. pastoris cloned in pDM 148 was about 500 bp in length while the 3'-UTR of MOXl from P. pastoris cloned into pDM 148 was about 1000 bp long.
• the cDNA insert was isolated from pARC 0770 by digestion with EcoRI and BflmHI (approximately 2.2 kb DNA fragment) and cloned between the EcoRI and B ⁇ mHI sites in pDM
• the resulting construct pARC 5771 (NCIMB 40721) contained the P. pastoris MOXl 5'-UTR followed by the NSP-BSSL coding sequence followed by S. cerevisiae ARG4 gene sequence and 3'-UTR of MOXl gene of P. pastoris while the entire DNA segment from 5'-UTR of MOXl to the 3'-UTR of MOXl was cloned at the MCS of pSK-.
• 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 5 (methanol slow growing) phenotype. 1.4. Screening for transformants expressing BSSL
• a lipase plate assay method was developed. The procedure for preparing these plates was as follows: to a solution of 2% agarose (final), 10 x 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 upto 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.
• 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.
• 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 KP0 4 buffer, pH 6.0, 400 ⁇ g/1 biotin, and 2% glycerol) for 24 h at 30°C until the cultures reached A -QQ close to 40.
• the induced cultures were incubated at 30°C with shaking for 120 h.
• 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'-MOXl, approximately 1000 bp segment of the alcohol oxidase promoter and a unique EcoRI cloning site.
• HIS4 P. pastoris histidinol dehydrogenase gene 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'-MOXl DNA was fused at the 3'-end of 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 at the unique N el site between HIS4 and 3'-MOXl region at 3' of the HIS4 gene.
• the plasmid pARC 5799 was digested with BglH 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 + .
• 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.
• 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].
• 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.
• Methanol feed rate was 6 ⁇ l/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
• 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 cone, of 0.25% and 0.5% respectively.
• BSSL enzyme activity in cell free broth increased from 40-70 mg/1 (equivalent of native protein) in 24 h to a maximum 200-227.0 mg/1
• the P. pastoris clone GS115[pARC 5799] was grown and induced in the fermenter as described in Section 3.1.
• BSSL 250 ml of culture medium (induced for 90 h) was spun at 12,000 x 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.
• 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 ul 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.
• 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.
• the cDNA of the fusion gene was further subcloned into P. pastoris expression vector pDM 148 (described in Section 1.2) by releasing the appropriate 1.8 kb fragment by EcoRI and BflmHI digestion of pARC 0788 and subcloning the fragment into pDM 148 digested with EcoRI and BamHI.
• the resulting construct pARC 5790 was digested with B ⁇ HI and a double stranded oligonucleotide linker of the physical structure BflmHI— EcoRI— BflmHI 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.
• 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.
• 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 BSSL-C. 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.
• 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.
• TELEPHONE +46-8-553 260 00
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• TELEX 19237 astra s
• GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG CCA Z ⁇ Z CCT GGC TGG 303 Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gin Pro :-:_. Pro Gly Trp 40 45 50
• 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 6 0
• MOLECULE TYPE protein
• HYPOTHETICAL NO
• NCIMB National Collections of Industrial and Marine Bacteria Limited
• NCIMB National Collections of Industrial and Marine Bacteria Limited
• NCIMB National Collections of Industrial and Marine Bacteria Limited

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