GB2217332A - Expression of salmon growth hormone in methylotrophic yeast of genus pichia - Google Patents

Abstract

22kD salmon growth hormone (SGH) is obtained in enhanced yield from methylotrophic yeasts of the genus Pichia which have been transformed with a vector comprising the sGH gene.

Description

EXPRESSION OF SALMON GROWTH HORMONE IN METHYIOTROPHIC YEAST OF THE GENUS PICHIA This invention relates to the field of recombinant DNA biotechnology. In one aspect, this invention relates to a process for expression of salmon growth hormone (sGH) in methylotrophic yeast of the genus Pichia. In another aspect the present invention relates to a novel yeast strain transformed with novel vectors containing a DNA sequence coding for the salmon growth hormone.

Salmon growth hormone (sGH) has been isolated and cloned in E.

col1 by Sekine et al. (Proc. Natl. cad. Sci. 82, (1985) 4306-4310). The said produced in E. coli was shown to be biologically active in stimulating the growth of rainbow trout.

The applications of this hormone in fish feed and baits for commercial aquaculture are far reaching. Several commercially important varieties of fish such as salmon and trout are responsive to this hormone and will exhibit increased growth when treated with sGH.

Unfortunately, E. coli has proven to be an unsuitable host for the production of sGH. For example, E. coli produces endotoxins which tend to contaminate the sGH produced. These endotoxins are undesirable contaminants which must be removed by expensive purification steps.

Additionally, the predominate 25 kD sGH protein produced in E. coli is in an inactive form which must be solubilized and treated to denature and then denature the protein. E. coli also only produces a limited amount of the more desirable biologically active 22kD sGH protein, tending instead to produce almost exclusively the inactive 25kD sGH protein.

Attempts have also been made to produce sGH in Saccharomyces cerevisiae such as is disclosed in European Patent Application 87104321.2.

However, the only form of protein expressed was the inactive 25kD sGH protein.

In accordance with the present invention, we have discovered in comparison with the prior art processes, an enhanced production of 22kD sGH protein is obtained from methylotrophic yeast strains of the genus Pichia which have been transformed with a compatible vector containing the sGH structural gene contained within an expression cassette.

In accordance with one aspect of the present invention, therefore, there is provided a process for the enhanced production of the 22kD sGH in methylotrophic yeast strains of the genus Pichia.

In accordance with another aspect, there are provided novel vectors containing a DNA sequence that codes for sGH.

In yet another aspect, there are provided novel linear integrative site-specific vectors containing a DNA sequence that codes for sGH.

Also, there are provided novel methylotrophic yeast strains of the genus Pichia transformed with a vector or vectors capable of the enhanced production of the 22kD sGH which are suitable for commercial fermentation.

The invention will be further described with reference to the accompanying drawings, in which: Figure 1(a) provides a representation of the plasmid pUC18 and the universal cloning site which was digested with EcoRI and HindIII in preparation for its insertion into the EcoRI site of pA0804.

Figure 1(b) provides a representation of pAû804 which will be digested with EcoRI and have the polylinker fragment from pUC18 inserted therein.

Figure 1(c) provides a represenation pf pHIL201 which is the product of the insertion of the pUC18 polylinker fragment into the EcoRI site of pA0804, all the restriction sites on the EcoRI fragment are not shown in this Figure although present.

Figure 2(a) provides a representation of psGHIB2 which will be digested with HindIII.

Figure 2(b) provides a representation of pHIL201 which will be cleaved at the SmaI site for the insertion of the HindIII fragment containing the sGH structural gene, all the restriction sites on the EcoRI fragment are not shown in this Figure although present.

Figure 2(c) provides a respresentation of pHILS1 which is the product of pHIL201 with the sGH structural gene inserted at the SmaI site. This plasmid will then be used as linear vector by partially digesting this plasmid with by II.

Referring now to the various aspects of the invention in more detail,7 the the gene coding for s(sH may be recovered directly from salmon according to the techniques used by Sekine et al. Proc. Natl. Acad.

Sci. Vol 82, pp. 4306-4310, July 1985. Other strategies employing recimbinant DNA technology could also be used to isolate this gene, such as artificial construction of the nucleotide sequences. However, for the practice of this invention it is preferable to isolate sGH gene from the plasmids deposited in connection with U.S. Patent 4,689,402 namely plasmid pSGHl, E. coli ESGH1 (FERM BP-551) deposited on June 23, 1984; pSGH14, E. coli ESGH14 (FERM BP-611) deposited on September 20, 1984; pSGHIB2, E. coli ESGHIB2 (FERM BP-612) deposited on September 20, 1984; pSGHIIB9 (FERM BP-707) deposited on February 8, 1985; and pSGHIiC2, E.

coli ESGHIIC2 (FERM BP-708) deposited on February 8, 1985. All deposits were made with FRI.

Isolation of the sGH gene from the plasmids above can be accomplished by any suitable technique or combination of techniques. For example, after cuituring an E. coli strain containing pSGHIIC2 the plasmid DNA could be recovered by using the method of Birmboin and Doby, Nucl. Acid Res. 7 (1979) 1513-1523. The plasmid could then be purified utilizing CsCl-EtBr density centrifugation. And finally the gene could be recovered by endonuclease digestion with HindIII and XbaI, gel electrophoresis, and electroeluton of the smaller HindIII-XbaI fragment containing the sGH gene.

Culturing the E. coli strain listed above may be accomplished by any suitable means. Generai techniques for culturing E. coli are already known in the art and any adaptation of these methods to the specific requirements of the strains containing the sGH gene is well within the abilities of those skilled in the art.

Recovery of plasmid DNA from E. coli can be accomplished by several techniques due to its compact size and closed spherical superhelical form. For example following the harvest, host cells may be pelleted by centrifugation and then resuspended and lysed. The lysate should be centrifuged to remove cell debris and the supernatant containing DNA retained. A phenol extraction can then be performed to remove most other contaminants from the DNA. The phenol-extracted DNA may then be further treated using a density gradient centrifugation or a gel filtration technique to separate the plasmid DNA from the bacterial DNA. The techniques for achieving the separation alluded to above are well known in the art and numerous methods of performing these techniques are known.

Sublease digestion of the plasmid containing the sGH gene may be accomplished by choosing appropriate endonucleases which will cut the plasmid selected in such a way as to facilitate the recovery of the intact sGH gene. The endonucleases used will depend on the source from which the sGH gene is to be excised. For example, the sGH gene contained in piasmid pscHIIC2 could be recovered as a HindIIi-YbaI fragment. Gel electrophoresis or another suitable technique would be necessary to separate it from the other frgment present.

Gel electrophoresis of DNA may be accomplished using numerous techniques. See P. G. Sealy and E. M. Southern, Gel Electrophoresls of SucLelc .Acids - A Practical Approach (D. Rickwood and B. D. Hames, eds.) p. 39 (i982). Elution may also be accomplished using numerous techniques appropriate for the gel involved, such as, electroelution, diffusion, gel dissolution (agarose gels) or physical extrusion (agarose gels). It is additionally recognized that elution may not be necessary with some gels such as high-quality, low melting temperature agarose.

Once the fragment containing the sGH gene is isolated, the fragment may require additional manipulation before it is inserted in the vector. These manipulations may include, but are not limited to the addition of linkers or blunt-ending the fragment.

Following the isolation of the sGH gene, the gene is inserted into a suitable vector such as a plasmid or linear transformation vector which is compatible with Pichia.

Plasmids have long been one of the basic elements employed in recombinant DNA technology. Plasmids are circular extrachromosomal double-stranded DNA found in microorganisms. Plasmids have been found to occur in single or multiple copies per cell. Included in plasmid DNA is the information required for plasmid reproduction, i.e. an autonomous replication sequence or ARS (an origin of replication may be included for bacterial replication). One or more means of phenotypically selecting the plasmid in transformed cells may also be included in the information encoded in the plasmid. Phenotypic or selection markers, such as antibiotic resistance or genes which compliment defective host biochemical pathways, permit clones of the host cells which have been transformed to be recognized, selected, and maintained.

To express the sGH gene in Pichia, the gene must be operably linked to a Pichia compatible- regulatory region and 3' termination sequence which forms the expression cassette which will be inserted into the host via a vector.

The following terms are defined herein for the purpose of clarification.

sSH--poLyTetide resulting from expression of the sGH gene.

Operably linked--refers to a juxtaposition wherein the components are configured so as to perform their function.

Regu'atory region--refers to heterogeneous DNA sequences which response to various stimuLi and affect the frequency of aRN.A transcription.

3' Termination sequence--sequences 3' to the stop codon which function to stabilize the mRNA such as sequences which elicit polyadenylation or stem loop structures.

"Pichia compatible' refers to DNA sequences which will perform their normal function in Pichia such as regulatory regions and 3 termination sequences derived from Pichia or closely related yeasts such as Sacchromvces cerevisiae and Hansenula polmmorpha.

Preferred for the practice of the present invention are integrative vectors, such as the linear site-specific integra.ive vector of Cregg as described in European Application Serial Number 86114700.7.

Such vectors comprise at least 1) a first insertable DNA fragment; 2) a selectable marker gene; and 3) a second insertable DNA fragment.

The first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of species of the genus Pichia.

The various components of the integrative vector are serially arranged forming a linear fragment of DNA such that the expression cassette and the selectable marker gene are positioned between the 3' end of the first insertable DNA fragment and the 5' end of the second insertable DNA fragment. The first and second insertable DNA fragments are oriented with respect to one another in the serially arranged linear fragment as they are so oriented in the genome of Pichia.

Nucleotide sequences useful as the first and second insertable DNA fragments are nucleotide sequences which are homologous with separate portions of the native Pichia genomic site at which genomic modification is to occur. Thus, for example, if genomic modification is to occur at the locus of the alcohol oxidase gene, the first and second insertable DNA fragments employed will be sequences homologous with separate portions of the alcohol oxidase gene locus. For genomic modification in accordance with the present invention to occur, the two insertable DNA fragments must be oriented with respect to one another in the linear fragment in the same relative orientation as they exist in the Pichia genome.Exemplary examples of nucleotide sequences which could be used as first and second insertable DNA fragments are nucleotde sequences selected from the group consisting of the alcohol oxidase (ilX1) gene, dihydroxyacetone synthase (DHrlS1) gene, p40 gene and HIS: gene. The AOXl gene, DHAS1 gene, p40 gene and HIS4 gene are described in European Patent Application 0 183 071.

The first insertable DNA fragment may contain an operable regulatory region which may comprise the regulatory region utilized in the expression cassette. The use of the first insertable DNA fragment as the regulatory region for an expression cassette is a preferred embodiment of this invention. Figure 2(c) provides a diagram of a vector utilizing the first insertable DNA fragment as a regulatory region for a cassette.

Optionally as shown in Figure 2(c) an insertion site or sites and a 3' termination sequence may be placed immediately 3' to the first insertable DNA fragment. This conformation of the linear site-specific integrative vector has the additional advantage of providing a ready site for insertion of a structural gene without necessitating the addition of a compatible 3' termination sequence.

The numerous cleavage sites as provided in the pHIL201 vector between the first insertable sequence and the 3' termination sequence are also advantageous in that fewer llnkers will be required to insert the structural gene into the vector.

It is also necessary to include at least one selectable marker gene in the DNA used to transform the host strain. This facilitates selection and isolation of those organisms which have incorporated the transforming DNA. The marker gene confers a phenotypic trait to the transformed organism which the host did not have, e.g., restoration of the ability to produce a specific amino acid where the untransformed host strain has a defect in the specific amino acid biosynthetic pathway or resistance to antibiotics and the like.

Exemplary selectable marker genes may be selected from the group consisting of the HIS4 gene and the ARG4 gene from Pichia pastoris and Saccharomvces cerevisiae, the invertase gene (sly2) from Saccharomvces cerevisiae, the G1S phosphotransferase gene from the E.

coli transposable elements Tn601 or Tn903.

Those of skill in the art recognize that additional DNA sequences can also be incorporated into the vectors employed in the practice of the present invention, such as for example, bacterial plasmid DNA, bacteriophage DNA, and the like. Such sequences enable the amplification and maintenance of these vectors in bacterial hosts.

If the first insertable DNA fragment does not contain a regulatory region, a suitable Plcnia compatible regulatory region will need to be inserted operably linked to the structural gene, in order to provide an operable expression cassette. Similarly if no 3' termination sequence is provided at the insertion site to complete the expression cassette, a 3' termination sequence will have to be operably linked to the structural gene to be inserted.

Those skilled in the art are aware of numerous regulatory regions which have been characterized and could be employed in conjunction with the Pichia genus. Exemplary Pichia compatible regulatory regions include but are not limited to yeast regulatory regions selected from the group consisting of acid phosphatase, galactokinase, alcohol dehydrogenase, cytochrome c, alpha-mating factor and glyceraldehyde 3-phosphate dehydrogenase regulatory regions isolated from Saccharomyces cerevisiae; the primary alcohol oxidase (.-iOX1), dihydroxyacetone synthase (DHASI), the p40 regulatory regions, and the HIS4 regulatory region derived from Pichia pastoris and the like.

Presently preferred regulatory regions employed in the practice of the present invention are those characterized by their ability to respond to methanol-containing media, such regulatory regions selected from the group consisting of AOXl, DHAS1, p40 and HIS4 disclosed in co-pending European Patent~Application 0 183 071.

The most preferred regulatory region for the practice of this invention is the AOX1 regulatory region.

3' termination sequences may be utilized in the expression cassette or be part of the vector as discussed above. 3' termination sequences may function to terminate, polyadenylate andíor stabilize the messenger RNA coded for by the structural gene when operably linked to r gene. A few examples of illustrative sources for 3' termination sequences for the practice of this invention include but are not limited to the Saccharomyces cerevisiae, Hansenula polymorpha, and Pichia 3 termination sequences. Particularly preferred are those derived from Pichia pastoris such as those selected from the group consisting of the 3 termination sequences of .tOX1 gene, DHAS1 gene, p40 gene and HIS; gene.

For the practice of the current invention it is currently preferred to use linear transformation vectors such as the BglII fragments of the constructs shown in-Figure 1(b) and (c).

The insertion of the sGH gene into suitable vectors may be accomplished by any suitable technique which cleaves the vector chosen at an appropriate site or sites and results in at least one operable expression cassette containing the sGH gene being present in the vector.

Ligation of sGH gene may be accomplished by any appropriate ligation technique such as utilizing TS DNA ligase.

The initial selection, propagation, and optional amplification of the ligation mixture of the sGH gene and a vector is preferably performed by transforming the mixture into a bacterial host such as E.

coli (although the ligation mixture could be transformed directly into a yeast host). Suitable transformation techniques for E. coli are well known in the art. Additionally, selection markers and bacterial origins of replication necessary for the maintenance of a vector in a bacterial host are also well known in the art.

The isolation and/or purification of the desired plasmid containing the sGH gene in an expression system may be accomplished by any suitable means for the separation of plasmid DNA from the host DNA.

Similarly the vectors formed by ligation may be tested preferably after propagation to verify the presence of the sGH gene and its operable linkage to a regulatory region and a 3' termination sequence. This may be accomplished by a variety of techniques including but not limited to endonuclease digestion, gel electrophoresis, or endonuclease digestion-Southern hybridization.

Transformation of plasmids or linear vectors into yeast hosts may be accomplished by suitable transformation techniques including but not limited to those taught by Hinnen et al, Proc. natal. acid. Sci. 75, (19i8) 19=9; Ito et al, J. Bacteriol 153, (1983) 163; Cregg et al Mol.

Cell Biol. 5 (1985) pg. 3376; or Sreekrishna et al, Gene, 59 (1987) pg. 115. Preferably for the practice of this invention are the transformation techniques of Cregg or Sreekrishna. It is deslrsble for the practice of this invention to utilize an excess of linear vectors and select for multiple insertions by Southern hybridization.

The yeast host for transformation may be any suitable methylotrophic Pichia host. Presently preferred Pichia hosts are auxotrophic Pichia strains such as GSllS (NRRL Y-15851). Although it is recognized~that wild type strains may be employed with equal success if SUC2 or a suitable antibiotic resistance marker is employed such as G18.

Transformed Pichia cells can be selected for using appropriate techniques including but not limited to culturing previously auxotrophic cells after transformation in the absence of a biochemical product required (due to the cells auxotrophy), selection by the detection of a new phenotype ("methanol slow"), or culturing in the presence of an antibiotic which is toxic to Pichia in the absence of a resistance gene contained in the transformant.

Isolated transformed Pichia cells are cultured by appropriate fermentation techniques such as shake flasks fermentation or the high density fermentation techniques as taught by U.S. Patent 4,414,329.

Expression may be accomplished by methods appropriate to the regulatory region employed. Preferably if methanol responsive regulatory regions are utilized, the induction of expression may be accomplished by the technique outlined in Example V.

Following expression of the sGH gene the presence of both the 22kD and the 25kD protein were detected by a Western blot. Total sGH protein production was estimated to be from about 1% to about 3% of the total cell protein. The percent of total sGH which was in the 22kD form was estimated to be about 5* to 20% of the total sGH present as determined by the modified Western blot.

The following non-limiting examples are provided to further illustrate this invention.

Examples General information pertinent to the Examples: Strains Pichia pastoris GS115 (his4) [NRRL Y-15851] was the host yeast strain used in these examples.

E. coli DG75' (hsdl, leu-6, lacY, thr-l, supE4, tonal lambda [-i) or JM107 (endAl, gyrA96, thil, hsdR 17 SupE44, relA, lambda (-), > (lac-Pro.AB), [F , traD36, ProAB, lacIqZAM151] were used for plasmid constructions as well as for propagatlon of plasmid DNAs.

Buffers, and Solutions, and Media The buffers and solutions employed in the following examples have the compositions given below: 1M Tris buffer 121.1 g Tris base in 800 mL of H20; adjust pH to the desired value by adding concentra;ed (35%) aqueous HC1; allow solution to cool to room temperature before final pH adjustment, dilute to a final volume of iL.

TE buffer 1.0 mM EDTA in 0.01 M (pH 7.4) Tris buffer SED 1 M sorbitol 2.5 mM EDTA 50 mM DTT --adjust to pH 8 SCE I M sorbitol 10 mM Sodium citrate 1 mM EDTA --pH to 5.8 with HCl CaS 1 M sorbitol 10mM CaCL2 10 mM Tris-HCl (pH 7.5j --filter sterilize PEG Solution 20% polyethylene glycol-3350 10 mM CaCl2 10 mM Tris-HCl (pH 7.4) --filter sterilize Minimal Fermenter Feed/FM-21 10% carbon source H3PO4 85% 3.5 ml/l CaSO4-2H2O 0.15 g/l K2SO4 2.38 g/l MgSO4.7H2O 1.95 g/l KCH 0.65 g/l YTM-4 1 ml/l SOS 1M sorbitol 10mM CaCl2 33.5% YEPD Breaking Buffer 50 mM Na3PO4, pH 7.5 5% glycerol 1 mM EDTA 1 mM PMSF (Sigma) YPD, 1 liter 10 g Yeast extract 20 g peptone 10 g dextrose SDR, 1 liter 13.4 g YNB 400 mg biotin # g biotin 182g Sorbitol 10 g dextrose 10 g agar 50 mg each of glutamine, methionine, lysine, leucine and isoleucine 2 g histidine assay mix SDHR, 1 liter SDR + 400 mg histidine KDHR - KDR + histidine (400 mg).

FM-21/YTM-54 Salts Medium H3PO4 (75%) 15.75 ml CaSO 2H20 0.15 g 60 g K2S04 9.50 g MgSO4-7H2O 1.95 g 7.i5 g KOH 2.60 g FeS047H20 .030 g CuS045H20 .00375 g ZnSO4.7H2O .0125 g MnSO4 H20 .001875 g Biotin .0005 g KI .0005 g Na2MoO4 .00125 g H3BO3 .000125 g IM3 Salts Medium KH2PO4 15.0 g K2HPO4 1.0 g MgSO4.7H2O 0.50 g CaSO4.2H2O 0.04 g (SH4)2S04 3.00 g Biotin 0.00005 g pH to 5.4 1 L water Example I Creation of pHIL201 The pA0804 plasmid is available in an E. coli host from the Northern Regional Research Center of the United States Department of Agriculture, Peoria, Illinois (accession number B-18021).Plasmid pHiL201 was constructed starting from pA0804 as follows: pA0804 (about4 pg) was digested to completion in two aliquots with 20 units oE EcoRI restriction enzyme in 25 pl of high salt buffer (10 mM magnesium chloride, 50 mM Tris-HCl-pH 7.5, lmM dithiothreitot, 100mM sodium chloride and 100 g/ml bovine serum albumin). The digestion was carried out for 1.5 h at 370 C. This mixture was then heated at 700 C for 6 minutes in a water bath and then transferred to an ice water bath. The DNA present in solution was precipitated by adding 3 pl of 3M sodium acetate and 80 pl of absolute ethanol. DNA precipitation was facilitated by incubation at -20 C for 135 minutes. The DNA precipitate was collected by centrifugation for 30 min. at 40 C in a microfuge.The DNA pellet was dried under vacuum. Each DNA pellet was dissolved in i8 pl of alkaline phosphatase buffer (50 mM Tris-HC1, pH 9.0 and lmM magnesium chloride) and incubated with 0.5 units of calf intestinal alkaline phosphatase for 30 minutes at 370 C. An additional 0.5 units of alkaline phosphatase was added, and incubation for 30 minutes at 370 C was repeated. Following this the reaction mixture was extracted three times with a phenol-chloroform mixture, two times with chloroform and four times with 500 pl of ether. The residual ether was evaporated in vacuum.

The aliquots were combined and the DNA was precipitated as described above. The DNA pellet was dissolved in 20 pl of water and stored at -20 C until further use. This DNA was designated as 'vector" Plasmid pUCl8 (about 10 pg) was digested with Bglf (20 units) and HindIII (10 units) in 20 L of high salt buffer (described above) at 370 C for 1 hour. BglI digestion helps to reduce the background due to regeneration of pUC18 during ligation. Sodium chloride and Tris were added to bring buffer concentration to high salt conditions described above and EcoRI (20 units) was added to release the universal cloning site by incubation at 370 C for 60 minutes. After digestion the DNA was precipitated as described above.The DNA precipitate was dissolved in 20 pl of water and stored at -20 C until later use. This DNA was designated as 'passenger".

Vector and passenger DNAs were mixed at a ratio of 1:2.5 (w/w) in 20 pl of DNA ligation buffer (50mM Tirs-HCl-pH 7.4, 10 mM magnesium chloride, 10mm dithiothreitol, 1" ATP and 100 pg/ml bovine serum albumin) at a final DNA concentration of 70 pg/ml and incubated overnight at 4 C and 2 units of T4DNA ligase. One half of the iigated sample was used to transform E. coli strain JM107 musing the method of Dagert and Ehrlich (Gene 6, 23 (1979). Several ampicillin resistant clones were obtained and they were tested on minimal media with ampicillin, X-gal and IPTG. A mixture of white and blue colonies were obtained. The blue colonies represent pUC18 background. Thus, the use of JM107 as a host allowed us to eliminate pUC18 containing background clones. Several white clones were subjected to small-scale lysis and EcoRI-BamHI restriction analysis which resulted in identification of clones containing pHIL201 (see Figure 1(c)). Large scale plasmid preparation was made from one such clone to obtain a sufficient quantity of pHIL201 for the next step (see Example III).

Example II Recovery of sGH Gene from psGHIB2 The sGH gene corresponding to about 0.9 Kb (the nucleotide composition of this fragment is indicated below) was released from psGHIB2 by HindIII digestion. About 10 pg of plasmid psGHIB2 (described in Sekine et. al., Proc. Natl. Acad. Sci. USA. vol. 82, pp. 4306-4310, July 1985) was digested at 370 C for 4 hours with 10 units of HindIII in 40 pl of medium salt buffer (10 mM Tris-HCl-pH 7.5, 10 mM magnesium chloride, 1 mM dithiothreitol, 50 mM sodium chloride and ' pg bovine serum albumin). Following digestion, 8 p1 (about 2 pg DNA) of the reaction mixture was electrophoresed on 0.9% agarose. Greater than 50 of the plasmid was found to be cleaved by HindIII under these conditions.

Gel electrophoretic patterns clearly indicated the presence of a DNA fragment of approximately 0.9 Kb corresponding to the sGH gene.

The cohesive ends of the DNA fragments resulting from HindIII cleavage of psCHIB2 were blunt ended using Klenow fragment of E. coli DNA polymerise I as follows: 32 pl of HindIII cleaved psGHIB2 (approximately 8 pg DNA) was mixed with 2.5 pl of dNTP mix (a mixture of 2 mM each of dATP, dTTP, dCTP and dGTP), 5 pl of lOx nick translation buffer (0.5 M Tris-HCl-pH 7.2, 0.1 M magnesium sulfate, 1 mM dithiothreitol and 00 g/ml bovine serum albumin), 10 pl double distilled water and 0.5 l (19 units) of Klenow fragment of DNA polymerase I.The reaction mixture was incubated at 220 C for 30 minutes and the reaction was terminated bv heating to 700 C for 5 minutes. Following this the whole sample was electrophoresed on 0.9% agarose gel and the portion of the gel containing the DNA fragment corresponding to the sGH gene (approximately 0.9 Kb) was excised from the rest of the gel and the DNA present in the gel piece was electroeluted in a dialysis bag into 400 pl of 5 mn EDTA (pH 8.0). The eluted DNA was phenol-chloroform extracted, precipitated, washed, dried and dissolved in 50 pl of double distilled water as described in the Example I above. The yield of the sGH DNA fragment was about 1 pg. This was used as the source of sGH gene fragment for the construction of pHIL51 (see Example III).

Table 1: Nucleotide sequence of the sGH gene fragment released by HindIII cleavage of psGHIB2. The initiator codon AIG and the translational termination codon TAG have been underlined in Table I.

The nucleotides indicated in lower case letters represent the portion filled in using Klenow fragment of DNA polymerase I to obtain blunt ended sGH gene fragment.

TABLE I AGCTTATG ATA GAA AAC CAA CGG CTC TTC AAC ATC GCG GTC AGT CGG GTG tcgaATAC TAT CTT TTG GTT GCC GAG AAG TTG TAG CGC CAG TCA GCC CAC CAA CAT CTC CAC CTA TTG GCT CAG AAA ATG TTC AAT GAC TTT GAC GGT GTT GTA GAG GTG GAT AAC CGA GTC TTT TAC AAG TTA CTG AAA CTG CCA ACC CTG TTG CCT GAT GAA CGC AGA CAG CTG AAC AAG ATA TTC CTG CTG TGG GAC AAC GGA CTA CTT GCG TCT GTC GAC TTG TTC TAT AAG GAC GAC GAC TTC TGT AAC TCT GAC TCC ATC GTG AGC CCA GTC GAC AAG CAC GAG CTG AAG ACA TTG AGA CTG AGG TAG CAC TCG GGT CAG CTG TTC GTG CTC ACT CAG AAG AGT TCA GTC CTG AAG CTG CTC CAC ATT TCT TTC CGT CTG TGA GTC TTC TCA AGT CAG GAC TTC GAC GAG GTG TAA- AGA AAG GCA GAC ATT GAA TCC TGG GAG TAC CCT AGC CAG ACC CTG ATC ATC TCC AAC AGC TAA CTT AGG ACC CTC ATG GGA TCG GTC TGG GAC TAG TAG AGG TTG TCG CTA ATG GTC AGA AAC GCC AAC CAG ATC TCT GAG AAG CTC AGC GAC CTC GAT TAC CAG TCT TTG CGG TTG GTC TAG TGA CTC TTC GAG TCG CTG GAG AAA GTG GGC ATC AAC CTG CTC ATC ACG GGG AGC CAG GAT GGC GTA CTA TTT CAC CCG TAG TTG GAC GAG TAG TGC CCC TCG GTC CTA CCG CAT GAT AGC CTG GAT GAC AAT GAC TCT CAG CAG CTG CCC CCC TAC GGG AAC TAC TCG GAC CIA CTG TTA CTG AGA GTC GTC GAC GGG GGG ATG CCC TIG ATG TAC CAG AAC CTG GGG GGC GAC GGA AAC GTC AGG AGG AAC TAC GAG TTG ATG GTC TTG GAC CCC CCG CTG CCT TTG CAG TCC TCC TTG ATG CTC .tAC TTG GCA TGC TTC AAG AAG GAC ATG CAC AAG GTC GAG ACC TAC CTG ACC AAC CGT ACG AAG TTC TTC CTG TAC GTG TTC CAG CTC TGG ATG GAC TGG GTC GCC AAG TGC AGG AAG TCA CTG GAG GCC AAC TGC ACT CTG TAG ACG CAG CGG TTC ACG TCC TTC AGT GAC CTC CGG TTG ACG TGA GAC ATC TGC TGG GCT GGA GAG GCA GCC AGC AAG TGC CTG TCT CCA GGG TTC GGT TTG ACC CGA CCT CTC CGT CGG TCG TTC ACG GAG AGA GGT CCC AAG CCA AAC CCA GAT ACA GAT TAG GCC TTG CCC TGC ACT GAG GTG CAT TIT CAA TTG GGT CTA TGT CTA ATC CGG AAC GGG ACG TGA CTC CAC GTA AAA GTT AAC AGA TTC TCC ATT GAA CAT GCT TTT CAG TCT GGA GTA ATT TAA TTT TGG TCT AAG AGG TAA CTT GTA CGA AAA GTC AGA CCT CAT TAA ATT AAA ACC ATC TGG TAG AGC CTG ACT CCA GGA GTT TTC AGG CAT TTG CAT TTT TTT TAG ACC ATC TCG GAC TGA GGT CCT CAA AAG TCC GTA AAC GTA AAA AAA CTC TGA ATC ACT CTG AGC TAC CAT TGA TTA GTA CAT TTA TAG AAA AGG GAG ACT TAG TGA GAC TCG ATG GTA ACT AAT CAT GTA AAT ATC TTT TCC TTA TTA AAT ATG CIA CTG TTT ATG CAT ACG TTA ATA TTT AGG GGT GAA AAT AAT TTA TAC GAT GAC AAA TAC GTA TGC AAT TAT AAA TCC CCA CTT ATG GGA ACT TGT AGA GCT CCA agct TAC CCT TGA ACA TCT CGA GGT TCGA Example III Creation of pHIL51 Plasmid The blunt ended sGH gene fragment from psGHIB2 obtained as described in Example II was inserted into SmaI (blunt end) site of pHIL201 plasmid (described in Example I) as follows: Plasmid pHIL201 (2 pg) in 20 pl of medium salt buffer (described in Example II) was cleaved with 5 units of Smal by incubation at 370 C for 2 hours.Following this, about 200 ng of the digested DNA was electrophoresed on 0.9% agarose gel which indicated that most of the DNA was cleaved by Smart. The Smal digested plasmid was treated with calf intentional alkaline phosphatase as described in Example I. The Smal cut and alkaline phosphatased pHIL201 was dissolved in 100 p1 water. For ligatlon, 100 ng of pHIL201 cleaved with SmaI and treated with alkaline phosphatase was mixed with 200 ng of blunt ended sGH gene fragment (Example II) in 20 pl volume of ligation buffer salt (described in Example I) and ligated overnight at 40 C with T4-DNA ligase. Following this, the ligation reaction was terminated by incubating at 650 C for 5 minutes. The ligated sample was digested with 5 units of SmaI for 30 minutes at 370 C as described before. The SmaI digestion after ligation reduces the interference in transformation by self-ligated pHIL201. The SmaI digested ligation mixture was used to transform E. coli DG75'.

Several ampicillin resistant clones were obtained and they were analyzed by alkaline lysis to identify the clones that contained the desired pHILS1 plasmid. Large scale plasmid preparation was conducted from one such clone and used for transformation of P. pastoris GS115.

Example IV Transformation of Pichia pastoris A. Vector preparation 100 pg of plasmid pHlL51 obtained from E. coli DG75 was subjected to a partial digestion with BglIl. A 6.1 Kb BglII fragment was obtained; see Figure 2(c). Because sGH contains an internal II site a complete digest could not be performed as this would result in the recovery of inoperative and incomplete integrative vectors.

The fragments obtained by the partial digest of pHILS1 were subjected to agarose gel electrophoresis. 1 pg of linear site-selective integrative sGH vectors contaminated with smaller BglII fragments was obtained by this process. This mixture of DNA fragments was used to transform Pichia pastoris strain GS115 (his4-) deposited with the Northern Regional Research Center of the U.S. Department of Agriculture, accession number NRRL Y-15851. Transformation with vectors containing the histidinol dehydrogenase gene will complement this defect in the histidine pathway, changing the GS11S transformed cells to Hiss.

Screening for transformants may therefore be easily accomplished by culturing the cells after transformation in a growth environment lacking histidine and recovering the cells capable of growing under this condition.

B. Cell Growth Pichia pastoris GS115 (NRRL Y-15851) was inoculated into about 10 ml of YPD medium and shake cultured at 300 C for 12-20 hours. 100 ml of YPD medium was inoculated with seed culture to give an OD600 of about 0.001. The medium was cultured in a shake flask at 300 C for about 12-20 hours &

The culture was harvested when the OD600 was about 0.2-0.3 (after approximately 16-20 hours) by centrifugation at 1500 g for 5 minutes using a Sorvall RCSC.

C. Preparation of Spheroplasts The cells were washed once in 10 ml of sterile water, and then centrifuged at 1500 g for 5 minutes. (Centrifugation is performed after each cell wash at 1500 g for 5 minutes using a Sorvall RT6000B unless otherwise indicated.) The cells were then washed once in 10 ml of freshly prepared SED, once in 10 ml of sterile 1M sorbitol, and finally resuspended in 10 ml of SCE buffer. 7.5 pl of 3 mg/ml Zymolyase (100,000 units/g obtained from Miles Laboratories) was added to the cell solution.

The cells were then incubated at 300 C for about 10 minutes. (.R reduction of 60% in ODpoo can be utilized as a correct time and concentration marker). The spheroplasts were washed once in 10 ml of sterile 1 sorbitol by centrifugation at 700 g for 5-10 minutes. (The time and speed for centrifugation may vary; centrifuge enough to pellet the spheroplasts but not so much they rupture from the force). 10 ml of sterile CaS was used as a final cell wash, and the cells were centrifuged again at 700 g for 5-10 minutes and resuspended in 0.6 ml of CaS.

D. Transformation GS115 cells were transformed with 1 pg of the linear sGH vectors using the spheroplast transformation technique of Sreekrishna et al. in Gene 59, 115-125 (1987). DNA samples were added (up to 20 p1 volume) to 12 x 75 mm sterile polypropylene tubes. (DNA should be in a suitable buffer, such as TE buffer); 100 pl of spheroplasts were added to each DNA sample and incubated at room temperature for about 20 minutes.

1 ml of PEG solution was added to each sample and incubated at room temperature for about 15 minutes and centrifuged at 700 g for 5-10 minutes. SOS (150 p1) was added to the pellet and incubated for 30 minutes at room temperature. Finally, 850 pl of 1 M sorbitol was added.

E. Regeneration of Spheroplasts A bottom agar layer of 20 ml of regeneration agar SDR was poured per plate at least 30 minutes before transformation samples were ready. In addition, 8 ml aliquots of regeneration agar were distributed to 15 ml conical bottom corning tubes in a 450 C bath during the period that transformation samples were in SOS. Aliquots of 50, 250 or 800 p1 of the transformed sample was added to the 8 ml aliquots of melted regeneration agar held at 450 C and poured onto plates containing the solid 20 ml bottom agar layer. The plates were incubated at 300 C for 3-5 days.

F. Selection of Transformants Transformants were selected for by culturing on SDR, a media lacking histidine. Cultures which grew in the absence of histidine were additionally screened for the "methanol slow" phenotype (indicating site selective integration). The transformed GS115 cells showing evidence of both phenotypes were then cultured and assayed for the production of sGH.

Pichia pastoris GSllS/pHILSl-62 was selected by this process and was deposited on March 29, 1988 in accordance with the Budapest Treaty with the Northern Regional Research Center in Peoria, Illinois, accession number SRRL(Y-18353).

Example V Expression of sGH A. Cell Culture Pichia pastoris GS115/pHIL51-69 was inoculated into two liters of IM-3 medium containing 2% glycerol at 300C. After this batch of glycerol was exhausted, continuous growth was established on F!-21 medium containing 1041 glycerol. The continuous culture was maintained at a dilution rate of 0.065 (Retention Time = 15.4 hr) until a cell density of approximately 50 g/l dry cell weight was obtained. After four reactor volumes of feed medium had been supplied to the fermentor the continuous stage of the fermentation was terminated. The production of salmon growth hormone was induced and maintained by the repeated injection of methanol at 0.25 to 0.5X of the culture volume such that the concentration of methanol was maintained between 0.1 and 0.8%. Samples were collected for analysis of salmon growth hormone at 0, 24, 50 and 73 hours of exposure of the culture to methanol. At 73 hours of methanol exposure, the culture was harvested by centrifugation. Approximately 300 g of wet cell paste was obtained from the remaining culture volume of 1.5 1. This corresponded to 199 g/l wet cell paste, or approximately 50 g/l dry cell weight, indicating that the cell mass did not significantly decrease during the methanol induction stage of fermentation.

B. Detection of sGH 10 mls of cells with an OD600 of 5-10 per ml were obtained from the fermentor. The cells were centrifuged at 5000 rpms using a Sorvall RT6000B refrigerated centrifuge. The pellet was washed with 1 mi of breaking buffer, centrifuged as above and resuspended in 0.5 ml breaking buffer. The mixture was vortexed for a total of 8 minutes, 2 to 4 minutes at a time, using one-half volume of acid-washed glass beads (Sigma, 450 micron). The mixture was centrifuged again at 12,000 rpms for 5 minutes using a microfuge to pellet the cell debris. The resulting pellet was resuspended in 200 l of the following solution: 10 nb Tris-Cl pH 8, 1.0 rnfl EDTA, 2.5 SDS, 5% ss-mercaptoethanol, and 0.01% BPB (bromophenol blue).For detection of sGH, this solution was run on a 15% SDS polyacrylamide gel for approximately 25 minutes. Standards used were purified .5 kD sGH and a Biorad low molecular weight standard. Protein was visualized on the gels by Coomassie Blue staining. Approximately 1-3% of the total cell protein was sGH, with 5-20; of this being the active 22kD form. Detection of the presence of the active 22 kD and the 25 kD species of sGH was performed by a modified Western using a triple antibody detection procedure: sGH mouse MoAb/anti mouse goat Ab/anti goat rabbit alkaline phosphatase. Both bands were visualized on the gels.

The examples provided are merely to illustrate the practice of the invention and should not be viewed as a limitation on the scope of our invention or appended claims. Reasonable variations and modifications, not departing from the essence and spirit of the invention are contemplated to be within the scope of the patent protection desired and sought.

Claims (21)

1. A process for the production of 22kD sGH protein which comprises culturing a transformed methylotrophic yeast strain of the genus Pichia transformed with at least one vector having a Pichia compatible expression cassette containing an sGH structural gene therein operably linked to a regulatory region and to a 3' termination sequence; allowing the product 22kD sGH protein to accumulate in the culture medium and thereafter recovering the product 22kD sGH protein from the culture.

2. A process according to claim 1, wherein there is used a yeast strain containing as said vector a plasmid or linear integrative site-specific vector containing said sGH expression cassette.

3. A process according to claim 2, wherein there is used a yeast strain containing as said vector a linear integrative site-specific vector containing the following serial arrangement; (a) a first insertable DNA fragment; (b) a marker gene, and at least one Pichia compatible expression cassette containing an sGH structural gene operably linked to a regulatory region and to a 3' termination sequence; and (c) a second insertable DNA fragment; wherein the order of the marker gene and cassette of component (b) may be interchanged.

4. A process according to claim 3, wherein the first and second insertable DNA fragments are derived from the DNA sequence of an AOX1, p40, DHAS or HIS4 gene isolated from Pichia pastoris.

5. A process according to claim 3 or 4, wherein the expression cassette in (b) comprises: (a) a regu;tory region comprising an AOX1, p40, DHAS or HIS4 regulatory region isolated from Pichia pastor is, or an acid phosphatase, galactosidase, alcohol dehydrogenase, cytochrome c, alpha-mating factor or glyceraldehyde 3-phosphate dehydrogenase regulatory region isolated from Saccharomyces cerevisiae; (b) an sGH structural gene that codes for sGH operably linked to said regulatory region and to (c) a 3' termination sequence isolated from the AOX1, p40, DHAS or HIS4 gene of Pichia pastoris.

6. A process according to claim 3, 4 or 5, wherein said marker gene is an HIS4 or ARG4 gene isolated from Pichia pastoris, a SUC2 gene isolated from Saccharomyces cerevisiae, or a G418R gene of bacterial Tn903 or Tn601.

7. A process according to claim 3, wherein said vector comprises (a) a first insertable DNA fragment which is about one kilobase of the 5' AOX1 regulatory region isolated from Pichia pastoris operably linked to (b) an sGH structural gene operably linked to (c) the 3' termination sequence of AOX1 isolated from Pichia pastoris ligated to (d) a marker gene which is HIS4 isolated from Pichia pastoris ligated to (e) a second insertable DNA fragment which is about 0.65 kilobases of the 3' AOX1 termination sequence.

8. A linear integrative site-specific vector comprising the following serial arrangement (a) a first insertable DNA fragment, (b) a marker gene and at least one Pichia compatible expression cassette containing an sGH structural gene operably linked to a regulatory region and to a 3' termination sequence, and (c) a second insertable DNA fragment.

wherein the order of the marker gene and cassette of component (b) may be interchanged.

9. A vector according to claim 8, wherein the first and second insertable DNA fragments are derived from the DNA sequences of an AOX1, p40, DHAS or HIS4 gene isolated from Pichia pastoris.

10. A vector according to claim 8 or 9, wherein the expression cassette in (b) comprises: (a) a regulatory region comprising an AOX1, p40, DHAS or HIS4 regulatory region isolated from Pichia pastoris, or an acid phosphatase, galactosidase, alcohol dehydrogenase, cytochrome c, alpha-mating factor or glyceraldehyde 3-phosphate dehydrogenase regulatory region isolated from Saccharomyces cerevisiae; (b) an sGH structural gene that codes for sGH operably linked to said regulatory region and to (c) a 3' termination sequence isolated from the AOX1, p40, DHAS or HIS4 gene isolated from Pichia pastoris.

11. A vector according to claim 9 or 10, wherein said marker gene is an HIS4 or ARG4 gene isolated from Pichia pastoris; an SUC2 gene isolated from Saccharomyces cerevisiae; or a G418R gene of bacterial Tn903 or Tn601.

12. A vector according to claim 8, comprising (a) a first insertable DNA fragment which is an operable regulatory region of the 5' AOX1 gene being about one kilobase in length isolated from Pichia pastoris operably linked to (b) an sGH structural gene operably linked to (c) the 3' termination sequence of AOX1 isolated from Pichia pastor is ligated to (d) a marker gene which is HIS4 isolated from Pichia pastoris ligated to (e) a second insertable DNA fragment which is about 0.65 kilobases of the 3' AOX1 termination sequence.

13. The vector pHILSi.

14. Methylotrophic yeasts of the genus Pichia transformed with a vector containing a Pichia compatible expression cassette comprising a regulatory region operably linked to an sGH gene operably linked to a 3' termination sequence.

15. Methylotrophic yeasts according to claim 14 which are of the species Pichia pastoris.

16. A methylotropic yeast according to claim 15, wherein the yeast is Pichia pastoris GS115.

17. Methylotrophic yeasts according to claim 14, 15 or 16, transformed with a vector as claimed in any one of claims 8 to 13.

18. Pichia pastoris GS115 transformed with the linear BqlII/BqlII sitespecific integrative vector cleaved from vector pHIL51.

19. Pichia pastoris GS115/pHIL51-62 NRRL(Y-18353).

20. A process for the production of 22kD sGH protein substantially as hereinbefore described in Example V.

21. A process for the transformation of Pichia pastoris substantially as hereinbefore described in Example IV.