METHOD FOR THE PRODUCTION OF BOVINE GROWTH HORMONE USING A SYNTHETIC GENE

Abstract: Synthetic steroids which are used to promote the growth of cattle and elevate the efficiency of feed remain in the body for a long time after ingestion and may have a detrimental effect when such products are consumed by humans. The invention relates to a method for the production of bovine growth hormone which is used to promote the growth of cattle, to increase the secretion of milk in cows and to elevate the efficiency of feed using a synthetic gene in yeast or E.coli. It has been found that bovine growth hormone may be produced economically and in large quantity in yeast or E.coli using gene manipulation technology.

Description: METHOD tOK THE PKODUCTION OF BOVINE GKOV/TH HOK NE USING A SYNTHETIC GENE BACKGROUND OF THE INVENTION The present invention relates to a method for the production of bovine growth hormone by synthetic gene in yeast or E.coli. Bovine growth hormone is used to promote the growth of cattle, to increase the secretion of milk in cows and to elevate tht efficiency of feed. Synthetic steroids, for example, Estradiol-compudose by Eli Lilly U.S.A. ; Estradiol benzoate-Synovax by Syntax, U.S.A. have been used in order to promote the growth of cattle and elevate the efficiency of feed in the livestock business up to now. But it has been found in developed nations including the U.S.A. that synthetic steroids remain in the body for a long time after they have been ingested with eed and then have a detrimental influence when such products are consumed by humans, and prohibition of their usage has increased. On the other hand, because bovine growth hormone does not remain inside the body after being ingested and it has a species- specificity, as it is a naturally occuring bovine protein, it is a desirable material for human beings. However, until early 1980's it has not been readily available to the livestock industry since it is extracted only from bovine pituitary glands and its amount has been largely limited for general use. The present inventors have discovered that bovine growth hormone may be produced economically and in large quantity in yeast or E.coli by using gene manipulation and accomplished the present invention SUMMARY OF- THE INVENTION One object of the present invention is to provide a method for the production of bovine growth hormone by means of using yeast as a host for expression vector. The other object of the present invention is to provide a method for the production of bovine growth hormone by means of using E.coli as a host for expression vector. Firstly, the method for production of bovine growth hormone by means of yeast as a host for expression vector comprises ; (a) synthesizing oligonucleotides having Sacl, Pstl and Sail restriction sites based on amino acid sequence of bovine growth hormone, (b) cloning one fragment with the Pstl/Sall sites and the other fragment with the Sacl/Pstl sites accoixling to the ligation strategy to a vector for E.coli(pUClS) comprising Pstl, Sacl and Sail restriction sites, respectively, (c) recloning two cloned fragments cut with Pstl/Sall and Pstl/Sacl restriction enzymes to the vector, pUC18, to get bovine growth hormone gene lacking a portion of the N-terminus, (d) inserting the partial bovine growth hormone gene and the N-terminal synthetic adaptor to a vector for E.coli consisting of a promoter and a terminator to make a cassette of promoter-bovine growth hormone gene comprising an initiation amino acid codon-terminator, (e) inserting the cassette to a yeast expression vector and (f) expressing the resultant vector in yeast cells. The amino acid codon of the N-terminal synthetic adaptor has the following base sequence of 58 mer and 50 mer with Ncoi and Sacl restriction sites : Ncoi site 3' Sacl site Secondly, the method for the production of bovine growth hormone by using E.coli as a host for expression vector comprises ; (a) synthesizing oligonucleotides with Sacl, Pstl and Sail restriction sites based on amino acid sequence of bovine growth hormone, (b) cloning the N-ter_G_Lnal fragment with the Sacl/Pstl sites and the C-teiminal fragment with the Pstl/Sall sites according to the ligatid strategy to a vector for E.coli(pUC18), respectively r (c) recloning the two cloned fragments cut with Pstl/SacI and Pstl/ Sail restriction enzymes to the vector, pUCIS, to get bovine growth hormone gene lacking a portion of the N-terminus, (d) inserting the partial bovine growth hormone gene and the synthetic adaptor to a vector for E.coli to obtain the bovine growth hormone gene ligated with the synthetic adaptor, Te) inserting the obtained bovine growth hormone gene to E.coli expression vector being able to express salmon growth hormone under the control of a trp promoter and (f) expressing the bovine growth hormone in E.coli. The synthetic adaptor designed to consists of a terminatirLg codon, TAA and an independent SI) sequence before the initiation codon, ATG and prevents the formation of the secondary structure of m-RNA between the SD sequence and the initiation codon ATG. The adaptor sequence comprises and upper strand of 73 mer (5'-CATG GAG GAA TTA TAA ATG GCT TIT CCG GCT ATG TCT CTA TCT GGT CTA TTC GCT AAC GCT GTT CTT CGA GCT-3' ) and a lower strand of 65 mer (3'-CTC CTT AAT ATT TAG CGA AAA GGC CGA TAC AGA GAT AGA CCA GAT AAG CGA TTG CGA CAA GAA GC-5' ) . The Sacl/Sail fragment of the bovine growth hormone gene of 526 bp and the synthetic adaptor were cloned to vector pSOD [Hallewell, R.A. et al, Nucleic Acid Res. 13, 2017 (1985)] treated with Ncol and Sail restriction enzymes to produce the bovine growth hormone gene ligated with the synthetic adaptor (pSOD BGH) . The vector (pSOD BGH) was treated with Ncol and Sail restriction enzymes to separate the bovine growth hormone gene and the separated fragment was inserted to an E.coli expression vector in which a part of the salmon growth hormone gene is removed by Xbai and Sail restriction enzymes, ptrp322HSGH [it is prepared by cloning salmon growth hormone to ptrp322 (Pharmacia Picataway, N.J. 08854, U.S-A; Russel, D.R. & Bennet- G.N. Gene 20, 231(1981)) and applied to Korea by the present applicant on the same date], to produce ptrphs BGH 1-13. The SD sequence and the initiation codon, ATG, the first part of the salmon growth hormone gene were used for expression in E.coli, in which the salmon growth hormone gene of 37 bp from the initiation codon and synthetic adaptor were ligated to bovine growth hormone gene. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows synthetic oligonucleotides corresponding to a whole bovine growth hormone gene and represented by the base sequence from the 5'-end to the 3'-end. Fig. 2 is a ligation strategy of oligonucleotides in accordance with Fig 1. Fig. 3 is a schematic cloning process of synthetic oligonucleotides to a vector for E.coli. Fig. 4 is the base sequence and putative amino acid sequence of the confirmed bovine growth hormone gene. Fig. δ is a cloning process to a vector for E.coli (pBS 100) and a vector for yeast expression (pCl/1) to produce boving growth hormone in yeast. Fig. 6 is a reassociation process of a complete bovine growth hormone gene to a vector for expression in E.coli. Fig. 7 shows the results confirmed by SDS-polyacrylamide gel elect- rophoresis of bovine growth hormone produced in yeast cells. Fig. 8 shows the results confirmed by SDS-polyacrylamide gel electrophoresis of bovine growth hormone produced in E.coli. DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT The present inventors have altered the 5'-end of the base sequence of bovine growth hormone by selecting amino acid codons preferentially used in yeast cells, based upon the amino acid sequence of bovine growth hormone, reported by Milter et al. [J.Biol. Chem. 255 (1980), 7521] and the total gene was chemically synthesized with a gene synthesizer (Applied Biosyste . Model 380B, USA) according to phosphoramidate's method in order that the NH2- terminal of the mature bovine growth hormone in the natural state is initiated with ala(Li, C.H. & Ash, L.J. Biol. Chem 203, 419-424). The synthetic oligonucleotides were ligated and cloned to a vecotr for E.coli, pUC18 [Norrander, J. et al. Gene 36 (1983) 101-106] to produce pBGH(526) comprising Sall/Sacl restriction fragment of 526 bp (Refer to Fig. 3). Firstly, a method for the production of bovine growth hormone from yeast cells is as follows ; The clone comprising the bovine growth hormone gene was produced and the Sacl/Sall fragment of 526 bp obtained from pBGH(526) with a synthetic adaptor was ligated between the Ncol and Sail sites of a vector having a promoter and a terminator gene, pBSlOO [Yeast 2.72 (1986) ; Chiron Corp. Erαeryvill, CA 94608, U.S.A] to express in yeast, wherein the adaptor chemically synthesized is comprised of the Ncol restriction site, an initiation codon and an amino acid codon corresponding to the N-terminal region. The resultant vector is pBS-BGH (576) (Refer to Fig. 5). pBS-BGH<576) was treated with BamHI to separate the BamHI fragment (about 2.7Kb) comprising a promoter, the bovine growth hormone gene and a terminator. The BamHI fragment was inserted into the BamHI restriction site of a yeast expression vector, pCl/1, which is automatically replicated in E.coli and yeast [ATCC 37115 . Brake et al. Proc. Natl. Acad. Sci USA 81 (1984), 4642] to produce pCl/1-BGH (Refer to Fig. 5). The above-mentioned DMA is cloned to yeast strain DC04 [Broach, J.R. & Hicks, J.B. Cell 21, 501(1980) ; Yeast Genetic Stock Center] by means of Hinnen et al's method. The transformed yeast cell was cultured in YEPD medium containing 2% glucose as in Example 4 for 48hrs. Whenever the concentration of glucose is exhausted, the bovine growth hormone is automatically induced and about 500mg bovine growth hormone per liter culture can be produced. Secondly, a method for production of bovine growth hormone in E.coli is as follows ; Vector pBGH (526) comprising 526 bp and a specifically designed adaptor were cloned to Ncol and Sail restriction sites of vector pSOD to obtain pSOD BGH containing a complete bovine growth hormone gene (Refer to Fig. 6). In order for bovine growth hormone to be expressed in E.coli, an E.coli expression vector, ptrp322H SGH was cloned and the polycistron method using an expression vector, ptrp322H SGH where salmon growth hormone is mass-expressed, is specifically adopted. The SD(Shine-Dalga no)sequence, an initiation codon (ATG) in the left side of the salmon growth hormone gene and the region including Xbal restriction site located at the right side from the initiation codon were used. The second SD sequence, the terminating codon (TAA. for stopping synthesis of a part of the salmon growth hormone and the complete bovine growth hormone gene were ligated thereto to maxi ize the expression of bovine growth hormone. The characteristic of the above method is as follows ; the " inhibition effect which is a result of the formation of a secondary structure of mRNA between the SD sequence and ATG, reported sis a problem in case that a foreign protein is expressed in E.coli, was eliminated by using the established gene construction whose ejφression rate was high. E.coli W3110 (ATCC 27325, Rockville, MD 20852, U.S.A.) containing an expression vector, ptrphs BGH 1-13 prepared as described above was selected and cultured under the condition of expression. By SDS polyacrylamide gel electrophoresis and densitomer scanning, it was confirmed that more than 30% of the whole E.coli proteins was bovine growth hormone, and also it was proven that it was bovine growth hormone by Western-blotting. The invention is illustrated by the following Examples, without them limiting its range. Example 1 : Ligation and cloning of synthetic oligonucleotides for bovine growth hormone gene ] The oligonucleotides (U7-U13/L7-L14) corresponding to the CCOH-terminal half of the whole synthetic oligonucleotides were taken the amount of 0.05 OD∑βo respectively, and then separately dried. Four units of T4 polynucleoti'de kinase were added to the total volume of 30 * 1 in the presence of buffer solution containing 50mM Tris-HCl (pH 7.5), ImM ATP, ImM DTT and lOmM MgCl∑ and each reaction mixture was incubated for 30mins. at 37 ° C in order to phosphorylate the 5'-end residue of the oligonucleotides. After all of the oligonucleotides were pooled and treated with an equal volumes of phenol and chloroform mixture, they were precipi¬ tated with ethanol. The precipitates were dissolved in 53 J> 1 of buffer solution containing 60mM Tris-HCl (pH 7.5), ImM DTT and lOmM MgCla. The solution was placed in 95 ° C water bath and kept at room temperature for 6 hrs. so that its temperature droped slowly. T4 DNA ligase (20 units) and 5 _P 1 of lOmM ATP are added herein and the 5'-and 3'-ends of oligonucleotides were ligated at room temperature for 10 mins. The above solution was treated with phenol and chloroform mixture and precipitated with ethanol. Ten units each of Pstl and Sail restriction enzymes were added to the precipitate in the presence of buffer'solution contaύiing 60π_M Tris-HCl (pH 7.6) and lOmM NaCl and reacted at 37 ° C for 1 hr. After 7%.polyacrylamide gel electrophoresis, a band corresponding to 280-330 bp was cut from the gel. After electroelution, the solution was precipitated with ethanol and ' the precipitate was dissolved in 20 > 1 of distilled water. Three 1 of the precipitate and lOng of a vector, pUC18, cut with Pstl and Sail restriction enzymes were ligated in the presence of the ligation buffer containing 60mM Tris-HCl (pH 7.5), lOmM DTT, lOmM MgCl 2 , ImM ATP and 10 units of T4 DNA ligase at 14 J C for 16 hrs. E.coli JM 103 [BRL, U.S.A., Messing, J. Methods in Enzymology 101, 20-78 (1983)] competent cell was added to the ligation mixture and was transformed according to Hanahan's method [J. Mol. Biol 118, 557 (1983)] at 37 U C overnight. The recombinant clone containing p3'-BGH was ■ selected from the white clonies according to Birnboim and Doly's method [Nucleic Acid Res. 7, 1513 (1979)]. On the other hand, the oligonuclotides (U2-U7/L2-L7) corresponding to the 5'-end of the whole synthetic 1 oligonucleotides were ligated by the same method as mentioned above and cloned to pUC18 cut with Sacl and Pstl restriction enzymes to produce p5'-BGH by the method of Fig. 3. The DNA sequence of p3'-BGH and pδ'-BGG were confirmed by the method of Sanger's dideoxy sequencing [Proc. Natl. Acad. Sci. USA 74, 5473-5477 {1977)] (Refer to Fig. 4). The Pstl-Sall restriction fragment from p3'-BGH and the SacI-PstI restriction fragment from p5'-BGH were respectively separated from 1% agarose gel and ligated to a vector, pUC18, cut with Sacl and Sail restriction enzymes to produce pBGH (526) (Refer to Fig. 3) . Example 2 : Manipulation of s?.nthetιc bovine growth hormone gene for exorssion m yeast ceils The deficient part corresponding to the 19 amino acids of the 5 J -end of the complete bovine growth hormone gene m pBGH 5 (526) was synthesized by DNA synthesizer in order for the mature gene to be cloned and expressed in yeast cells. The Ncol restriction site, an initiation codon and nucleotides corresponding to 18 amino acids were synthesized by selecting codons preferentially used in yeast cells(Refer to Fig. 5). 10 The process for cloning is as follows:pBGH (526) was treated with Sacl and Sail restriction enzymes to obtain a 526 * bp restriction fragment. The separated fragment and the above-mentioned synthetic adaptor were inserted into Ncol and Sail restriction sites of pBSlϋO containing an inducible 15 promoter and a terminator [Chiron Corp. Emeryville, CA 94608, U.S.A. ; Yeast 2, 72 (1986)]. One J- 1 1 of each phosphorylated adaptor, 3.P 1 (30ns) of Sacl/Sail fragment of separated bovine growth hormone and 1J-" 1 (7ng! of vector pB lOO, cut x.ιth Ncol and Sail restriction _... enzymes were mixed, keot. at 65 ' C for 15 mins. ana cooled siowlv at room temperature. Two y 1 of lOmM ATP, 2_P 1 of 10-fold concentrated ligation reaction buffer, 20 units of ligase and 8 " 1 of distilled water were added and reacted at 14 a C for 16 hrs. As described in Example 1, the mixture was transformed to E.coli HB 101 (ATCC 33694) and pBS-BGH (576) comprising a promoter, the complete bovine growth hormone gene &nd a terminatior was produced (Refer to Fig. 5). The vector, pBSlOO, contains the hybrid promoter of promoter of alcohol dehydrogenase II and glyceraldehyde 3' -phosphate dehydrogenase and which can be induced whenever glucose is depleted. The pBS-BGH (576) was treated with BamHI restriction enzyme to separate the BamHI restriction fragment of about 2,700 base pairs, containing a hybrid promoter, the bovine growth hormone and a terminator of glyceraldehyde 3'-phosphate dehydrogenase. The BamHI restriction fragment was inserted into the BamHI restriction site of the expression vector pCl/1 (ATCC 37115), which is able to be replicated in yeast cell, to obtain pCl/1-BGH (Refer to Fig. 5). Ten " g of pCl/1-BGH DNA was protoplast-transformed to yeast strain DCC4 [Yeast Genetic Stock Center, University of California, Berkeley, CA, USA ; Broach, J.R. &. Hicks, J. B. Cell 21 501 (1980)] according to Hinnen's method [Proc. Natl, Acad. Sci. USA 75 1929, (1978)] and plated on agar- -plates without leucine, comprising 6.7g of Yeast Nitrogen Base without amino acids, 182g of sorbitol 2% glucose, 0.25g of Leu-deficient amino acid supplements and 20g of Bactoagar. per liter medium. After culture at 30 ° C for 5 days, transformants were obtained. Example 3 : Manipulation of bovine growth hormone gene to an E. coli expression vector Codons preferentially used in E.coli were selected between the Sacl restriction site of bovine growt hormone gene and an initiation codon ATG. A te__π_ination codon, TAA and independent SD sequence for a polycistronic construction with pSOD designed so as that the secondary structure of mRNA was not formed in the vicinity- of the SD sequence and initiation codon ATG. The adaptor were an upper strand of 73 mer (5'-CATG GAG GAA 17 TEA TAA ATG GCT TTT CCG GCT ATG TCT CTA TCT GGT CTA TTC GCT AAC GCT GTT CTT CGA GCT-3' ) and a lower strand ' Of 65 mer (3' CTC CTT AAT ATT TAC CGA AAA GGC CGA TAC AGA GAT AGA CCA GAT AAG CGA TTG CGA CAA GAA GC-5') synthesized by the DNA synthesizer. The synthetic adaptor and the Sacl-Sall restriction fragment of 526 bp obtained frora.pBGH(526) were inserted to a vector pSOD, cut with Ncol and Sail restriction enzymes to obtain the pSOD BGH containing the complete bovine growth hormone gene(Refer to Fig. 6). In order that the bovine growth hormone is mass-expressed in E.coli using the trp promoter, a polycistron method in which the complete bovine growth hormone gene is arranged behind the " NH2-terminal region of the salmon growth hormone gene mass- expressed in E.coli, were developed and executed as in Fig. 6. That is, 5 J J g of an expression vector, ptrp322H SGH DNA in which the salmon growth hormone is mass-expressed was reacted with 10 units of Xbal restriction enzyme in a buffer solution containing SOmM Tris -HC1 (pH 8.0), lOmM MgCl 2 and 50mM NaCl at 37 ° C for 1 hr. " The solution was treated with phenol and chloroform mixture and precipitated with ethanol. The precipitate was dried and dissolved in 15.5 y 1 of distilled water. Then dNTP (dGTP, dCTP, dATP, dTTP) was added so as that the final concentration in 20 J 1 of the total reactant became 250 y M and the solution was reacted with 2.5 units of Klenow enzyme in a buffer solution containing 50mM Tris-HCl <pH 8.0) , lOmM MgClz and 50mM NaCl at room temperature for 20mins. in order that the site, cut with Xbal restriction enzyme of salmon growth hormone gene was made blunt-end. After the activity of Klenow enzyme was inactivated by keeping the above solution at 95 ° C for 2 mins, 10 units- of Sail restriction enzyme, 6.5 y 1 of IM NaCl and 20.5 y 1 of distilled water were added and incubated at 37 ° C for 1 hr. A DNA fragment of 2.4 Kb was separated from 1% agarose gel. The electroeluted DNA was dissolved in 15 y 1 of TE comprising lOmM Tris-HCl (pH 7.5) and ImM EDTA. On the other hand, in order to obtain the DNA fragment of the complete bovine growth hormone gene and independent SD sequence, 10 y g of pSOD BGH DNA prepared by the above-mentioned method was rected with 12 units of Ncol restriction enzyme in a buffer solution comprising 150mM NaCl, 6mM Tris-HCl (pH 7.9) and 6mM MgCl∑ at 37 J C for 1 hr. The reactant was treated with phenol and chloroform mixture and precipitated with ethanol. The precipitate was dried and dissolved in 31.5 y 1 of distilled water. dNTP (dGTP, dCTP, dATP, dTTP) was added so as that the final concentration became 250 y M, and the solution was reacted with 2.5 units of Klenow enzyme in a bufer solution containing 50mM Tris-HCl (pH 8.0), lOmM MgCla and 50mM NaCl at room temper¬ ature for 20 mins. After the solution was kept at 95 ° C for 2mins, 20 units of Sail restriction enzyme, 7 J J 1 of IM NaCl and 10 y 1 of distilled water were added and then incubated at 37 ° C for 1 hr. The DNA fragment of 595 dp was isolated from 1% agarose gel electrophoresis, electroelution, and dissolved in 15 y 1 of TE. The lOng of vector DNA of 2.4 Kb obtained by treating ptrp 322 H SGH DNA with Xbal, Klenow enzyme and Sail restriction enzyme and δOng of DNA containing, the complete bovine growth hormone gene were ligated with 20 units of T4 DNA ligase in a buffer solution containing 60mM Tris-HCl (pH 7.5), ImM DTT 1 and lOmM MgCl 2 at 14 " C for 16hrs. Then it was cloned to E.coli HB101 according to Hanahan's method to obtain ptrphsBGH 1-13. The obtained vector, ptrphsBGH 1-13 was cloned to E.coli W 3110 (ATCC 273 ' 25). Example 4 Cultivation of yeast for producing bovine growth hormone and its identification Each 3ml of yeas y cells transformed with vector pCl/1- BGH was cultured in a medium without leucine (6.7g Yeast Nitrogen Base without amino acids, 0.25g of Leu-deficient amino acids supplements and 2% glucose per liter medium) at 30 °C for 24 hrs. YEPD culture medium (100 ml) comprising 2% peptone, 1% yeast extract -and 2% glucose was inoculated with 1ml of overnight culture, and further cultured at 30 ° C for 24 hrs. 40ml of ethanol was added thereto and the solution was further cultured for 24hrs. The resultant ODβso was about 40. That ODβso of 10 was taken, centrifuged., and dissolved in 400 y 1 of a buffer solution containing lGmM Tris-HCl (pH 7.5), ImM EDTA, 2mM phenyl methyl sulfonyl fluoride (PMSF) and 8M urea and glass beads having a diameter of 0.45 mm were added and vortexed vigorously. After rupturing the cell wall and allowing the bovine growth hormone to elute into the buffer solution, 4 y 1 of eluted solution was executed by electrophoresis on 12.5% SDS polyacrylamide gel. The results are represented in Fig. 7; Lane 1 represents molecular weight of marker proteins from BRL company. Lane 2 represents total proteins of yeast cell transformed with vector pCl/1-BGH before induction with ethanol. Lanes 3-8 represent total proteins of yeast cell transformed with vetor pCl/1-BGH after induction with ethanol. Lane 10 represents total proteins of yeast cells transformed with vector pCl/1 without bovine growth hormone gene. As seen in lane 3 of Fig. 7, the bovine growth hormone expressed at a band about 22Kd in an amount corresponding to 20% of the total proteins by a densitometric scanning of the protein gel. Example δ : Mass-expression of boving growth hormone in E.coli W 3110 and confirmation by SDS poly¬ acrylamide gel electrophoresis and Western- blotting E.coli W 3110 comprising the expression vector, ptrphs BGH 1-13 was cultured on LB medium with 40 y g/ml of ampicillin at 37 " C for 16 hrs with shaking, and at " the point that ODβso of 0.5, 60 y g/ml of IAA (Indole acrylic acid) was added. The solution was further cultured at 37 ° C for 18hrs. The cultured cells corresponding to ODβso of 1.0 was centrifuged and dissolved in 100 y 1 of Laeπmli sample buffer , solution [Laemmli U.K. Nature 227, 680 (1970)]. The solution was heated at 100 ° C for 5 mins. and then 10 y 1 of it was run to 12.5% SDS polyacrylamide gel electrophoresis. The results are represented in (A) of Fig 8 ; (A) represents the results confirmed by SDS-polyacryla__u.de gel after the expression strain of bovine growth homone was cultured under the conditions described above. Lane 1 represents the uniπduced E.coli W3110 with ptrphs BGH. Lanes 2-6 represent the induction by IAA. As shown in (A) of Fig. 8, a protein band of 22Kd which can not be seen in uninduced cells .appears to be an amount of more than 30% of total proteins of E.coli. Western-blotting was done by using monoclonal antibody against bovine growth hormone to which horse radish peroxidase conjugated [Accurate Chemical Scientific.Corp. U.S.A.] according to Burnette et al.'s method [Anal. Biochem. 112, 195 (1981)] and the results are represented in (B) of Fig 8. (B) represents the result confirmed by Western-blotting of bovine growth hormone expressed in E.coli. Lanes 1-3 represent the strain which does not contain bovine growth hormone gene. Lane 4 represents the uninduced cells. Lane 5 represents the induction by IAA. As seen in (B) of Fig. 8, the immunoactivity appears at the same location as the protein band which appears in molecular weight, 22 kilodaltons. That is, the present inventors confirmed that the bovine growth hormone expressed has i_mmunoactivity. M9 medium comprises 40mM K 2 HPO., 22mM KH 2 P0 , 8.5mM NaCl, 18.7mM NH4CI, ImM MgS0 , O.lmM CaCl 2 , 10y g/ml Vit Bi, 0.4% CAA(Casamino acid), 1% glucose and 40j J g/ml ampicillin.

Claims: CLAIMS 1. A method for the production of bovine growth hormone, comprising (a) synthesizing oligonucleotides according to amino acid sequence of bovine growth hormone, (b) cloning the oligonucleotides ligated by the ligation strategy to an E.coli vector to produce bovine growth hormone gene lacking a portion of the N-terminus, (c) inserting the bovine growth hormone gene and the N-terminal synthetic adaptor to a vector with a promoter and a terminator to make a cassette of promoter-bovine growth hormone gene comprising an initiation ami o acid codon-teπninater, (d) insertion the cassette to a yeast expression vector and (e) expressing the resultant vector in yeast cells. 2. A method for the production of bovine growth hormone to claim 1, wherein the synthetic oligonucleotides have Sacl, Pstl and Sail restriction sites. 3. A method for the production of bovine growth hormone according to claim 1, wherein the bovine growth hormone gene lacking a portion of the N-terminus is produced by cloning the C-terminal fragment with the Pstl/Sall sites and the N-terminal fragment with the SacI/PstI sites to a vector (pUC18) comprising Pstl, Sacl and Sail restriction sites, respectively. 4. A method for the production of bovine growth hormone according to claim 1, wherein the N-terminal synthetic adaptor is the following nucleotide sequence having Ncol and Sacl restriction sites. Ncol site ' SacI site 5. A method for the production of bovine growth hormone according to claim 1, wherein the resulting vector for expression in yeast is a vector pCl/1-BGH obtained by treating the yeast expression vector w th BamHI and then inserting the cassette of promoter-bovine growth hormone gene comprising initiation amino acid codon-terminator to it. 6. A method for the production of bovine growth hormone, comprising (a) synthesizing oligonucleotides having Sacl, Pstl and Sail restriction sites according to amino acid sequence of bovine growth hormone, (b) cloning the oligonucleotides ligated by the ligation strategy to E.coli vector to produce bovine growth hormone gene, (c) cloning the partial bovine growth hormone gene and synthetic adaptor to E.coli vector to obtain the bovine growth hormone gene ligated with the synthetic adaptor, (d) inserting the obtained bovine growth hormone gene to E.coli expression vector being able to express salmon growth hormone under the control of a trp promoter, and (e) expressing the resultant vector in E.coli 7. A method for the production of bovine growth hormone according to claim 6, wherein the synthetic adaptor with a terminating codon TAA and an independent SD sequence before an initiation codon, ATC of bovine growth hormone gene prevents the formation of the secondary structure of m-RNA between the SD sequence and the initiation codon ATG. 8. A method for the production of bovine growth hormone according to claim 6 or 7, wherein the synthetic adaptor comprises an upper strand of 73 mer (5'-CATG GAG GAA TTA TAA ATG GCT TTT CCG GCT ATG TCT CTA TCT GGT CTA TTC GCT AAC GCT GIT CTT CGA GCT-3' ) and a lower strand of βδmer (3'-CTC CTT AAT ATT TAC CGA AAA GGC GCA TAC AGA GAT AGA CCA GAT AAG CGA TTG CGA CAA GAA GC-5' ) 9. A method for the production of bovine growth hormone according to claim 6, wherein the bovine growth hormone gene ligated with the synthetic adaptor is obtained by cloning the Sacl-Sall fragment comprising 526 bp of bovine growth hormone gene and a synthetic adaptor to a vector pSOD, treated with Ncol and Sail restriction enzyines. 10. A method for the production of bovine growth hormone according to claim 6, wherein the resulting E.coli expression vector is a vector ptrphs BGH 1-13 produced by (a) treating vector pSOD BGH with Ncol and Sail restriction enzymes to separate the bovine growth hormone gene and (b) cloning the separated fragment to the E.coli expression vector, ptrp322H SGH in which a part of the salmon growth hormone was eliminated by treating Xbal and Sail restriction enzymes. 11. A method for the production of bovine growth hormone according to claim 6, wherein the expression in E.coli was executed by using SD sequence and an initiation codon ATG in front of the salmon growth hormone gene. 12. A method for the production of bovine growth, hoππone according to claim 6 or 11, wherein the expression in E.coli was executed by ligating the bovine growth hormone gene with a synthetic adaptor comprising the SD sequence and a terminating codon TAA to the ^ 2 -terminal region of salmon growth hormone gene, in order to make a polycistronic construction.

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