METHOD FOR THE PRODUCTION OF PORCINE GROWTH HORMONE USING A SYNTHETIC GENE IN YEAST CELLS

Abstract: High protein content feed or synthetic steroids are used to elevate the efficiency of feed and promote pig growth. However, the steroids are not metabolized quickly but remain in the body for a long time and may have a detrimental influence on humans. The invention relates to a method for the production of porcine growth hormone which improves the growth of pigs and the efficiency of feed using a synthetic gene in yeast cells. It has been found that porcine growth hormone may be produced economically and in bulk in yeast by gene manipulation technology.

Description: METHOD FOR THE PRODUCTION OF PORCINE GKOWTH HORMONE USING A SYNTHETIC GENE IN YEAST CELLS BACKGROUND OF THE INVENTION The present invention relates to a method for the production of porcine growth hormone by a synthetic gene in yeast cells to improve the growth rate of pigs and the efficiency of feed. Traditionally, pig breeders have used feed with a high protein content or synthetic steroids in order to promote growth and elevate the efficiency of feed. But because feed with steroid supplements is not metabolized quickly but remains in the body for a long time and has a detrimental influence on humans, developed nations are prohibiting its use. Taking this into account, the present inventors have discovered that porcine growth hormone may be produced economically and in bulk in yeast by gene manipulation technology and intend to apply this discovery to the pig breeding business. Therefore, they have accomplished this by discovering that as a result of using yeast cells, in a manner different from other processes previously known (Seeburg et al. DNA2{1983) , 37) , natural and mature porcine growth hormone in which the N-terminus is initiated from ala maybe mass-produced. SUMMARY OF THE INVENTION The object of" the present invention is to provide a method for the production of porcine growth hormone by using yeast as a host for expression vector. Namely, the method for the production of porcine growth hormone by using yeast as a host for expression vector comprises (a) synthesizing oligonucleotides with Sacl, Xbal and Sail restriction sites based on amino acid sequence of porcine growth hormone, (b) cloning the C-terminal Xbal/Sall fragment of the synthetic oligonucleotides ligated by the ligation strategy to an E.coli vector, pUC18, which is treated with Xbal/Sall restric¬ tion enzymes, (c) cloning the N-terminal Sacl/Xbal fragment of synthetic oligonucleotides treated with Sacl/Xbal restriction enzyme to the above cloned vector to produce the porcine growth hormone lacking a portion of the N-terminus, (d) cloning the porcine growth hormone gene and N-terminal synthetic adaptor to an E.coli vector comprising a promoter and a terminator to produce a cassette of a promoter-porcine growth hormone gene with an N- terminal synthetic adaptor-terminator, (e) reclonmg the cassette to a yeast expression vector and (f) expressing the resultant vector in yeast cells. The N-terminal synthetic adaptor gene has the following base sequence with Ncol and Sacl restriction sites ; 5' + CAIGGCGTCCCϋGCrA'l XIGC'lXiAGC 3' 3* CGC GGGCCGATACGGCGAC + 5' Ncol site Saci site The resulting expression vector is the vector (pCl/1-PGH) cloned of a cassette comprising the promoter-porcine growth hormone gene with an N-terminal synthetic adaptor-terminator, a complete 2 micron gene, a Leu 2d gene and an origin of replication. BRIEF DESCRlPrlON OF THE DRAWINGS Fi .1 shows synthetic oligonucleotides corresponding to the porcine growth hormone gene and represented by the base sequence from the 5'-end to the '-end. Fig. 2 is a ligation strategy of oligonucleotides. Fig. 3 is a schematic cloning strategy of synthetic oligonu¬ cleotides to a vector of E.coli (pUCl8). Fig. 4 is the base sequence and putative amino acid sequence of confirmed porcine growth hormone gene. Fig. 5 is a cloning process for a vector of E.coli(pPGAP) and a yeast expression vector (pCl/1) to produce porcine growth hormone in yeast. Fig.6 are the results confirmed by SDS-polyacrylamide gel electrophoresis of porcine growth hormone produced in yeast cells. DETATT.PD DESCRIPTION OF A PREFERRED EMBODIMENT The present inventors have selected the base sequence of porcine growth hormone gene with an amino acid codon preferentially used in yeast cells, based upon the amino acid sequence of porcine growth hormone reported by Seeburg et al. [DNA 2(1983) 37], and the total gene of mature porcine growth hormone in the natural state, in which the N-terminus initiates with ala, is chemically synthesized with a gene synthesizer (Applied Biosystem Model 380B, USA) according to the phosphoramidate-s method. The synthetic oligonucleotides are ligated as shown in Fig. 2 and cloned to an E.coli vector, pUCIS, [Norrander et al. Gene 36 (1983) 101-106] to produce a vector pPGH(552) comprising a Saci/Sall restriction fragment of 552 base pairs, which does not have an N- terminal region of a mature porcine growth hormone gene of 579 base pairs ( efer to Fi . 3) . In order to produce a clone comprising the complete porcine growth hormone gene and express it in a yeast host, the Saci/Sall fragment was separated from pPGH(552) and a synthetic adaptor was inserted between the Ncol site and the Sail site of the vector comprising a promoter and a terminator gene, pPGAP [Clontech Lab, Palo Alto, CA 94303, U.S.A. Holland, J.P. & Holland, J.J.J. of Biological Chemistry 255, 2596-2605 (1980), Bitter, G.A. & Egan, K.M. Gene 32, 263-274(1984) 20B-12, Zhu, X.L. et al., Mol. Gen. Genet 9194, 31-41(1984) ; ATCC 34025] to obtain pPGAP-PGH (579), wherein the adaptor chemically synthesized is comprised of an Ncol restriction site, an initiation codon and some amino acid codons corresponding to the N-terminal region (Refer to Example 3). pPGAP-PGH(579) is treated with BarnHI restriction enzyme to separate the BamHl fragment (about 1970 base pairs) comprising a promoter, the procine growth hormone gene and a terminator. The BamHl fragment is inserted into the BamHl restriction site of a yeast expression vector, pCl/1, which is automatically replicated in f coli and yeast [ATCC 37115 ; Brake et al., Proc. atl. Acad. Sci USA 81 (1984), 4642], to produce pCl/l-PGH(Re ' fer to Fig. 5). The expression vector is cloned to a yeast strain DC04 [Yeast Genetic Stock Center, Broach, J.R. & Hicks, J.B. Cell 21, 501 ( 1980)] by means of the method of Hinnen et al. The transformed yeast cell was cultured in YEPD medium comprising 2% glucose as in Example 5 for 24 hrs, porcine growth hormone was produced depending on growth rate of the cell and 200mg of pig growth hormone per liter of culture can be obtained at the ODeso = 20. The invention is illustrated by the following Examples, without them limiting its range. Example 1 : Ligation of synthetic oligonucleotides of * porcine growth hormone gene and cloning to vector pUClS In order to obtain the complete porcine growth hormone gene from the synthetic oligonucleotides having the gene sequences of Fig. 1, ligation strategy such as in Fig. 2 and vector pUC18 comprising Sacl, Sail, Xbal, etc. restriction sites were used. The oligonucleotides (U7-U14/L8-L14) corresponding to the 3'-end of the Xbal and Sail restriction fragments from synthetic oligonucleotides were collected at the amount that the OD260 of each oligonucleotide equals to 0.05 and then separately dried. Four units of T4 polynucleotide kinase were a ded to a total volume of 30^ 1 in the presence of a buffer solution comprising 50mM Tris-HCl(pH7.5), ImM ATP, ImM DTT and lOmM MgCla and they were reacted at 37 °C for 30 mins. to phosphorylate the 5'-end residue of the oligonucleotides. .After the oligonucleotides were pooled and treated with an equal volume of phenol and chloroform mixture, they were precipitated with ethanol. The precipitate was dissolved in 53 * 1 of a buffer solution comprising 60mM Tris-HCl (pH7.5), ImM DTT and lOm gcia. The solution was placed in a 95 ° C water bath and kept at room temperature for 6 hrs. so that as its temperature drops slowly, each oligonucleotide produced base pairing with complementary sequence. T4 DNA ligase (20 units) and δ " 1 of lOmM ATP were added and the 5'- and 3'-ends of the oligonucleotides were ligated at room temperature for lOπiins. The above solution was treated with phenol and chloroform mixture and precipitated with ethanol. Ten units each of Xbal and Sail restriction enzymes were added to the precipitated nucleic acid in the presence of a buffer solution comprising 60mM Tris(pH7.6) , lOmM Cl2 and lOOmM NaCl and reacted at 37 ° C for 1 hr. After 7% polyacrylamide gel electrophoresis of the above mixture, a band corresponding to 200-300 base pairs was cut from the gel. After electroelution, the precipitates were dissolved in 20 J-" 1 of distilled water. Three J 1 of DNA and lOng of a vector, pUC18, cut with Xbal and Sail restriction enzymes were ligated in the presence of the ligation solution comprising 60mM ϊris-HCl(pH7.5) , lOmM DTT, lOmM MgCla, ImM ATP and lOunits of T4 DNA ligase at 14 - C for 16hrs. E.coli M103[BRL, U.S.A., Messing, J. , Methods in lαizymology, 103, 20-78 (1983)] competent cell was added to the ligation reactant and transformed according to Hanahan's method [J.Mol. Biol 116, 557(1983)] at 37 ° C. The clone containing p3'-PGH was selected from the white colonies by using Birnboim and Doly's method fNucleic Acid Res. 7, 1513 (1979)]. On the other hand, the oligonucleotides (U1-U7/L2-L8) corresponding to the 5'-end Sacl and Xbal restriction fragment of the complete porcine growth hormone gene were ligated by the same method as mentioned above and cloned to the p3'-PGH vector cut with Sacl and Xbal restriction enzymes to produce pPGH(552) as shown in Fig. 3. The nucleotide sequence was confirmed by Sanger's dideoxy sequencing method [Proc. Natl. Acad. Sci. USA 74, 5473-5477] (Refer to Fig. 4). Example 2 : Manipulation of synthetic porcine growth hormone gene for expression in yeast cell pPGH(552) does not comprise 9 amino acids in the 5'-residue of a complete porcine growth hormone and in order to clone the complete porcine growth hormone gene and to have it expressed in yeast cells, the deficient part of the δ'-end was synthesized by the gene synthesizer. The adaptor comprising the Ncol restriction site, the initiation codon and the codon correspon- ding to 8 amino acids was synthesized by selecting codons prefe- rentially used in yeast cells and the Sacl restriction site was synthesized at the other end (Refer to Fig. 5). The process of cloning was as follows ; pPGH{552) was treated with Sacl and Sail restriction enzymes to obtain a restriction fragment corresponding to 552 base pairs and the fragment was separated through agarose gel eϊectrophoresis. The fragment and the synthetic adaptor were inserted between the Ncol and Sail - restriction sites of a vector, pPGAP, comprising a promoter and a terminator. The detailed procedures are as follows : The 5'-end of each synthetic adaptor was phosphorylated with T4 polynucleotide kinase as described in Example 1. One 1 of each phosphorylation solution, 3 - 1 1 (30ng) of the Saci/Sall fragment separated from pPGH(552) and 1J> 1 (7ng) of vector pPGAP cut with Ncol and Sail restriction enzymes were mixed and kept at 65 ° C for 15 mins. They were cooled slowly to room temperature. Two > 1 of lOmM ATP, 2±> 1 of a 10-fold concentrated ligation reaction buffer solution, ± 1 of ligase and 8J> 1 of distilled water were added and reacted at 14 ~ C for 16 hrs. Based upon the method as in Example 1, the above was cloned to E.coli cell HB101 [ATCC 37017] , and pPGAP-PGH comprising the promoter, the complete porcine growth hormone gene and the terminator for expression in yeast was produced. The vector, pPGAP, contains a glyceraldehyde-3'-phosphate dehydrogenase promoter, a constitutive promoter which is expressed according to the growth of the cell and a terminator. pPGAP-PGH was treated with BamHl restriction enzyme to separate the BamHl restriction fragment about 1,970 base pairs, coπφrising a glyceraldehyde-3'-phosphate dehydrogenase promoter, a porcine growth hormone gene and a glyceraldehyde-3'-phosphate dehydrogenase terminator. The BamHl restriction fragment was . inserted into the BamHl restriction site of the expression vector pCl/1 which can be replicated in yeast cells, to produce pCl/1-PGH (Refer to Fig. 5). The pCl/1-PGH gene was cloned to yeast strain DC04 according to Hinnen's method [Proc Natl. Acad. Sci, USA 75 (1978), 1929]. After culturing at 30 " C for 5 days, a recombinant clone with porcine growth hormone gene was picked and identified by the method such as in Example 3. Example 3 : Cultivation of yeast for producing porcine growth hormone and its identification Each 3ml of yeast cells transformed with vector pCl/1-PGH was cultured in a culture medium without leucine (6.7g of Yeast Nitrogen Base without amino acids, 0.25g of leu-deficient supplements and 6% glucose per 1 of culture medium) at 30 ° C for 24hrs. The culture was added to 100ml of YEPD culture medium comprising 2% peptone, 1% yeast extract and 2% glucose and cultured at 30 ° C for 24 hrs. At the ODβ5o equaled to around 40, the fraction corresponding to ODβ50 of 10 was collected and centrifuged. it is dissolved in 400J J 1 of a buffer solution containing lOmM Tris-HCKpH 7.5), ImM EDTA, 2mM phenyl methyl sulfonyl fluoride and 8M urea and glass beads, 0.45mm in diameter with the same volume, were added and shaken vigorously. After rupturing the cell wall and letting porcine growth hormone elute into the buffer solution, vM 1 of eluted solution was executed by electrophoresis on 12.5% SDS polyacrylamide gel. The results are represented in Fig. 6; Lane 8 represents the standard M. . of protein from the BioRad company. Lanes 1 and 5 represent total proteins of yeast cells transformed with the vector pCl/1 without porcine growth hormone gene. Lanes 2-4 represent total proteins of yeast cells transformed with the vector pCl/1-PGH containing the porcine growth hormone gene. As shown in lanes 2-4 of Fig 6, it is evident by gel scanning that porcine growth hormone appears at a band about 22Kd in an amount corresponding to 10% of the total protein. The amino acid sequence confirmed (Biomedical Resource Center, University of Calfornia, San Francisco) shows the mature porcine growth hormone starting from alanine and in which methionine might be processed by amino peptidase in vivo.

Claims: CLAIMS 1. A method for the production of porcine growth hormone, comprising (a) synthesizing oligonucleotides with Sacl, Xbal and Sail restriction sites and codons preferentially used in yeast based on amino acid sequence of porcine growth hormone, (b) cloning " the porcine growth hormone gene and N-terminal synthetic adaptor to an E.coli vector comprising a promoter and a terminator to produce a cassette of promoter-porcine growth hormone gene-terminator, (c) recloning the cassette to a yeast expression vector and (d) expressing the resultant vector in yeast cells. 2. A method for the production of porcine growth hormone according to claim 1, wherein the N-terminal synthetic adaptor has the following base sequence with Ncol and Sacl restriction sites; Ncol site Sacl site

Inventors: HAN KYU BEOM, JEONG HYEON HO, JO JUNG MYEONG, LEE TAE GYU, LEE TAE HO, LEE YONG BEOM, PARK YEONG WOO, HAN, KYU, BEOM, JEONG, HYEON, HO, JO, JUNG, MYEONG, LEE, TAE, GYU, LEE, TAE, HO, LEE, YONG, BEOM, PARK, YEONG, WOO
Applicants: LUCKY LTD, LUCKY, LTD.
Application year: 1987
Main classification: C12N15
IPC codes: C07K14, C12N15, C12P21, C12R1