AU713218B2 - Process for the mass production of bovine growth hormone - Google Patents

Process for the mass production of bovine growth hormone Download PDF

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AU713218B2
AU713218B2 AU40768/95A AU4076895A AU713218B2 AU 713218 B2 AU713218 B2 AU 713218B2 AU 40768/95 A AU40768/95 A AU 40768/95A AU 4076895 A AU4076895 A AU 4076895A AU 713218 B2 AU713218 B2 AU 713218B2
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Joong Myung Cho
Chun-Hyung Kim
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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/61Growth hormone [GH], i.e. somatotropin
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    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/36Vector systems having a special element relevant for transcription being a transcription termination element

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Description

AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT 4*
S
9 9.
*5 Name of Applicant: Actual Inventors: Address for Service: L G CHEMICAL LTD.
Chun-Hyung KIM Joong-Myung CHO CULLEN CO., Patent Trade Mark Attorneys, 240 Queen Street, Brisbane, Qld. 4000, Australia.
Invention Title: PROCESS FOR THE MASS PRODUCTION OF BOVINE GROWTH HORMONE The following statement is a full description of this invention, including the best method of performing it known to us la- PROCESS FOR THE MASS PRODUCTION OF BOVINE GROWTH HORMONE FIELD OF THE INVENTION The present invention relates to a process for the mass production of bovine growth hormone("BGH"). More specifically, it pertains to an expression vector comprising a modified BGH gene, wherein the nucleotide sequence at its 1 0 5'-end region is modified without changing the amino acid sequence encoded therein, and a trpA transcription terminator is inserted after the 3'-end of the modified BGH gene; an E.
coli cell transformed with the expression vector; and a process for the mass production of BGH which comprises culturing the E. coli transformant and separating BGH from the culture.
too*** BACKGROUND OF THE INVENTION Steroidal animal growth promoters such as Estradiol- Compudose(Eli Lilly), Estradiol Benzoate-Synovax(Syntax Agribusiness Inc.) and Zeramol-Ralgro(International Minerals and Chemicals) have been employed in stock breeding for increasing the feed efficiency and weight gain of the stock.
However, use of such steroidal growth promoters has become restricted since they are fat-soluble, and, consequently, have a long residence time in the stock tissue, which may 2 adversely affect humans when consumed.
In contrast, animal growth hormones, which are produced and secreted by animal pituitary glands, are not fat-soluble, show species-specificities and have been reported to enhance the growth and milk production of the stock and also to increase the feed efficiency(Bauman, D. et al., J. of Animal Sciences, 60, 583(1985); Hart, I. C. et al., J. of Endocrinology, 105, 189(1985); Newswatch, June 17, 1985; and Wall Street Journal, July 22, 1986)).
10 Since 1940's, natural animal growth hormones separated and purified from the pituitary glands of animals have been used for promoting the growth of animals, but their applications to stock breeding have been restricted by the limited supply of the hormones. Recent developments in 15 genetic technologies, however, make it possible to massproduce the animal growth hormones using microorganisms, E. coli.
P.H. Seeburg et al. have separated cDNA encoding BGH from the bovine pituitary gland; determined the nucleotide sequence of the cDNA and the amino acid sequence of BGH; cloned the cDNA into an E. coli expression vector comprising a trp promoter and expressed BGH in E. coli which is transformed with the vector(DNA, 2, 37(1983)).
Thereafter, many attempts have been made to increase the expression rate of BGH in E. coli.
For instance, H. J. George et al. have disclosed that the expression rate depends on the nucleotide sequence and 3 the length of the region between the ribosome binding site and the translation initiation codon, ATG; and produced
BGH
in an amount equivalent to 15% of the total proteins produced in E. coli(DNA, 4, 273(1985)).
Olsen et al. have produced BGH in an amount of 10 to on the basis of total proteins produced in an E. coli cell by culturing the cell transformed with an expression vector comprising a modified BGH gene, wherein the region between the ribosome binding site and the translation initiation 10 codon, ATG, is A-T rich, under the control of a trp promoter(J. Biotechnol., 9, 179(1989)).
Watson et al. have produced BGH in an amount of 20% on the basis of total proteins produced in an E. coli cell by culturing the cell transformed with a plasmid comprising a 15 modified BGH gene wherein one of the nucleotides encoding four amino acids in the N-terminal region of BGH is changed through random mutation(Gene, 86, 137(1990)).
It has also been published that a modified BGH gene is prepared by using the codons preferred by E. coli; constructed an expression vector for BGH by inserting the BGH gene into an E. coli vector comprising a salmon growth hormone("SGH") gene under the control of a trp promoter and ligating a synthetic linker between the SGH gene and BGH gene; and produced BGH in an amount of 27% on the basis of total proteins obtained by culturing an E. coli cell transformed with the vector(Korean Patent Publication No. 92- 3665).
4 However, the maximum amount of BGH that can be produced using processes of the prior art is limited to 30% on the basis of total proteins in E. coli.
On the other hand, a trpA transcription terminator has been reported to be a very efficient rho-independent terminator (Christie, G. E. et al., Proc. Natl. Acad. Sci.
U.S.A, 78, 4180(1981)), and, therefore, a desired protein may be mass-produced while repressing the production of undesirable proteins derived from the plasmid by inserting a 10 trpA transcription terminator after the 3'-end of the gene encoding the desired protein(Gentz, et al., Proc. Natl.
Acad. Sci. U.S.A, 78, 4936(1981)).
Matsuki et al. have produced rat calmodulin in an amount of 30% on the basis of total proteins in E. coli by culturing S 15 the cell transformed with a plasmid comprising a trp promoter, a calmodulin gene and a trpA transcription terminator(Biotech. Appl. Biochem., 12, 284(1990)).
Sato et al. have reported that, when interleukin-2 gene is expressed by employing a plasmid comprising an interleukin-2 gene under the control of a trp promoter and a trpA transcription terminator inserted after the 3'-end of the interleukin-2 gene, the amount of interleukin-2 becomes about 5 times higher than that produced by a control group wherein the trpA terminator is not inserted(J. Biochem., 101, 525(1987)).
Despite these developments, there has continued to exist a need to develop a process for the mass production of BGH in a commercially feasible and economical scale.
SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an expression vector which is suitable for the mass production of BGH.
Another object of the present invention is to provide a host cell transformed with the expression vector.
A further object of the present invention is to provide a process for the mass production of BGH, which comprises culturing the transformant.
In accordance with the present invention, there is provided an improved process for the mass production of BGH by constructing an expression vector comprising a modified BGH gene, wherein the nucleotide sequence at its 5'-end region is modified without changing the amino acid sequence encoded therein, and a trpA transcription terminator inserted after the 20 3'-end of the modified BGH gene; and producing BGH in an amount of more than 50% on the basis of the total protein obtained by culturing an E. coli cell transformed with said vector.
In a first embodiment, the invention provides an Sexpression vector for bovine growth hormone (BGH) comprising a 25 promoter, a BGH gene and a terminator, said vector being characterised in that a part of a salmon growth hormone gene contained in a SacI/PvuI fragment of PCR-amplified plasmid ptrphs BGH 1-13 herein defined is present between said promoter a a S" and the 5'-end of the BGH gene; the terminator is trpA S3u< transcription terminator inserted no more than 50 nucleotides downstream of the stop codon of said BGH gene; and the nucleotide sequence at the 5'-end region of the BGH gene is modified for minimizing the formation of a secondary structure in mRNA and increasing the expression rate, without changing the amino acid sequence encoded therein.
In a second embodiment, the invention provides an E. coli cell transformed with the expression vector of the first embodiment.
In a third embodiment, the invention provides a process for the mass production of bovine growth hormone, which comprises culturing the E. coli transformant of the second embodiment and separating bovine growth hormone from the culture.
BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description oA a
A
6 of the invention, when taken in conjunction with the accompanying drawings, in which: Fig. 1 compares the nucleotide sequences at the regions of a BGH gene(ptrphs BGH 1-13) and of its variant (ptrphs BGHRAN Fig. 2 A depicts a schematic diagram for constructing plasmid ptrphs BGHRAN comprising a modified BGH gene; Fig. 2B displays a schematic diagram for constructing plasmids ptrp3H BGH 1-13 and ptrp3H BGHRAN comprising the BGH 10 gene or a variant thereof together with a trpA transcription terminator ligated at its 3'-end, respectively; Fig. 3 provides the result of SDS-polyacrylamide gel electrophoresis(PAGE) using the E. coli cell precipitates transformed with plasmid ptrphs BGH 1-13, ptrphs BGHRAN ptrp3H BGH1-13 or ptrp3H BGHRAN; and Fig. 4 presents the results of determining the amount of
S.
bovine growth hormone with a densitometer, which is expressed in the E. coli cells transformed with plasmids ptrphs BGH 1- 13, ptrphs BGHRAN ptrp3H BGH 1-13 and ptrp3H BGHRAN, respectively.
DETAILED DESCRIPTION OF THE INVENTION In accordance with one aspect of the present invention, there is provided an expression vector for the massproduction of BGH, which comprises a modified BGH gene and a trpA transcription terminator inserted after the 3'-end of 7the modified BGH gene.
The modified BGH gene of the present invention has a modified nucleotide sequence at its 5'-end region for minimizing the formation of a secondary structure in mRNA and increasing the expression rate, while encoding the unaltered amino acid sequence of normal bovine growth hormone. The modified BGH gene of the present invention preferably has at its 5'-end region the nucleotide sequence of: 1 0 5'-ATG GCT TTT CCG GCT ATG TCT CTA TCT GGC
C
TA TTC GCA AAT GCC GTT CTT CGA GCT CAG ~CAT CTT CAT CAG CTG GCT-3' wherein ATG is a translation initiation codon of BGH gene.
S 15 The expression vector of the present invention also comprises a trpA transcription terminator after the 3'-end of the modified BGH gene, which preferably has the nucleotide sequence of: 5'-AGCCCGCCTA ATGAGCGGGC TTTTTTTT-3' The expression vector of the present invention may be prepared in accordance with any conventional genetic recombination or gene synthesis method, a procedure described below.
First, random primers are synthesized based on the information on nucleotide sequences of a BGH gene(see Korean 8 Patent Publication No. 92-3665). These random primers are designed to introduce into BGH a recognition site for restriction enzyme SacI and to randomly modify the nucleotide sequence encoding BGH without altering the amino acid sequence encoded therein.
Also prepared is a primer which corresponds to a part of Amp' gene in recombinant vector ptrphs BGH 1-13 comprising the BGH gene(see Korean Patent Publication No. 92-3665;
ATCC
6897 5(deposited on May 6, 1992)) and has a recognition site 10 for restriction enzyme PvuI. Then, a PCR is carried out by using the above primers and plasmid ptrphs BGH 1-13 as a template to amplify a modified BGH gene. The amplified BGH gene is digested with restriction enzymes PvuI and SacI and ligated with a DNA fragment which is obtained by digesting 15 plasmid ptrphs BGH 1-13 with the same enzymes, to obtain an expression vector of BGH. The expression vector is used to .:.**transform E. coli, and a plasmid comprising the modified BGH gene is separated and named ptrphs BGHRAN.
S: Further, in order to introduce a trpA transcription terminator into the BGH expression vector, primers for PCR, which comprise a trpA transcription terminator gene, a recognition site for restriction enzyme SalI and another recognition site for restriction enzyme PvuI, are prepared.
Then, a PCR is carried out by using the above primers and plasmid ptrphs BGH 1-13 as a template to amplify a DNA fragment comprising the trpA transcription terminator. The amplified DNA fragment is digested with restriction enzymes 9 PvuI and SalI, and ligated with a DNA fragment which is obtained by digesting plasmid ptrphs BGHRAN with the same enzymes, to obtain an expression vector of BGH. The expression vector is used to transform E. coli, and a plasmid comprising the modified BGH gene and the trpA transcription terminator is separated and named ptrp3H BGHRAN.
The expression vectors of the present invention are used to transform an E. coli cell, E. coli W3110(ATCC 37339), which is suitable for expressing the BGH gene in the 10 expression vector. The transformed E. coli cell is cultured under a condition that allows the expression of BGH and the expressed BGH can be separated and purified from the culture in accordance with any conventional method.
Therefore, the present invention further provides a 15 process for the mass production of BGH, which comprises culturing the E. coli cells transformed with the expression vector of the present invention and separating BGH from the culture. In accordance with the inventive process, it is possible to produce BGH from the E. coli transformant in an amount of more than 50% of the total proteins produced.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.
10 Example 1: Amplification of a Modified BGH Gene and Construction of an Expression Vector Comprising Same (Step 1) Random primers for PCR, which correspond to the region of BGH and have the following nucleotide sequences, were synthesized on the basis of the information on 10 nucleotide sequences of BGH gene(see Korean Patent Publication No. 92-3665): Primer PBGHRAN: 15 NGGRAANGCCATTTATAATTCCTCCA-3' wherein N means A, T, G or C; and R means A or G.
Primer PBGHRAN comprises a SacI recognition site and modified nucleotide sequence encoding 16 amino acids from the N-terminal of BGH.
Further, primer PPBR3720 comprising a part of Ampr gene in plasmid ptrphs BGH 1-13(see Korean Patent Publication No.
92-3665; ATCC 68975) and a PvuI recognition site was also synthesized, the nucleotide sequence thereof being as follows: 5'-TCCTTCGGTCCTCCGATCGTTGTCA-3'.
11 (Step 2) To a test tube were added 1 ng of plasmid ptrphs BGH 1-13 as a template, 2 pg of each of primers PBGHRAN and PPBR3720 prepared in (Step 10 p2 of 10x polymerase reaction buffer(10 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgCl 2 gelatin), 10 pR of a mixture of dNTP's(2 mM each of dGTP, dATP, dCTP and dTTP), and 2.5 unit of Taq DNA polymerase(Perkin Elmer Cetus, and distilled water 10 was added thereto to a final volume of 100 pR.
The PCR was carried out by repeating 25 times the cycle of: 95 0 C for 1 min.(denaturation), 550C for sec.(annealing), and 72°C for 2 min.(polymerization).
The PCR product obtained above was subjected to 15 polyacrylamide gel electrophoresis and as a result, it was confirmed that about 990 bp of DNA was amplified. The DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment BGHRAN.
(Step 3) 2 pg of plasmid ptrphs BGH 1-13 was completely digested with SacI in NEB(New England Biolabs Inc.) buffer 1(10 mM Bis Tris propane-HC1, 10 mM MgCl 2 1 mM DTT, pH 7.0) at 37 0 C for 1 to 2 hours, and then completely digested with PvuI in NEB buffer 3(100 mM NaC1, 50 mM Tris-HCl, 10 mM MgCl 2 1 mM DTT, pH 7.9) under the same condition. The resulting mixture was 12 subjected to 0.7% agarose gel electrophoresis to isolate about a 2 kb fragment, which was named fragment
PBGH-T/P.
2 Vg of fragment BGHRAN obtained in (Step 2) was completely digested with SacI in NEB buffer 1, and then completely digested with PvuI in NEB buffer 3. The resulting DNA fragment was extracted with phenol/chloroform and then dissolved in 20 p. of TE buffer. The DNA fragment was named fragment BGHRAN-T/P.
A ligation reaction was carried out as follows by using 10 the DNA fragments obtained above. A reaction tube was provided with 100 ng of each of fragments BGHRAN-T/P and PBGH-T/P, 2 pi of 10x ligation buffer, and 10 units of T4 DNA ligase; and distilled water was added thereto to a final volume of 20 The ligation was carried out at 16 0 C for 12 15 hours.
E. coli W3110(ATCC 37339) was transformed with the S: igation mixture to obtain a recombinant E. coli transformant containing plasmid ptrphs BGHRAN comprising the fragment BGHRAN(see Fig. 2).
Example 2: Expression of the Modified BGH Gene and Sequencing of the Gene (Step 1) About 100 E. coli transformant colonies obtained in Example 1 were cultured in a liquid Luria medium(6% Bacto- .ii?;?7rl- I- -a~---~ricl 13 tryptone, 0.5% yeast extract, 1% NaCl) containing 50 pg/m£ of ampicillin at 37 0 C for 12 hours. 3 m£ of the culture was transferred into 300 m£ of M9 medium(40 mM K 2
HPO
4 22 mM
KH
2
PO
4 8.5 mM NaC1, 18.7 mM NH 4 Cl, 1% glucose, 0.1 mM MgSO 0.1 mM CaCI 2 0.4% casamino acid, 10 pg/m£ Vit. 40 pg/m£ ampicillin); and cultured with shaking at 37 0 C for 4 hours.
When the O.D. value of the culture at 650 nm reached about 0.3, indole acrylic acid(IAA) was added to the culture to a final concentration of 50 pg/mR. After 4 hours, the 10 O.D. of the resulting culture was determined and the culture was centrifuged at 11,000 rpm for 25 min. to collect the E.
coli cell precipitates. The cell precipitates were subjected to 15% SDS-PAGE by employing Laemmli's method(Nature, 227, 680(1970)) to confirm the expression of BGH. The clones S 15 showing a 40% higher amount of expressed BGH than the E. coli transformant comprising plasmid ptrphs BGH 1-13(control group) were selected and the plasmid contained therein was named ptrphs BGHRAN #5(see Fig. 3).
(Step 2) Sequencing of the BGH gene in plasmid ptrphs BGHRAN obtained in (Step 2) was carried out as follows. Plasmid ptrphs BGHRAN #5 was completely digested with SacI in NEB buffer 1, and then completely digested with EcoRI in NEB buffer 3. The reaction mixture was subjected to 7% polyacrylamide gel electrophoresis to separate a DNA fragment 14 having about 187 bp, which was named fragment BGHRAN-T/R.
On the other hand, 2 pg of plasmid M13mpl8 was completely digested with SacI in NEB buffer 1, and then, completely digested with EcoRI in NEB buffer 3. The resulting mixture was subjected to 0.7% agarose gel electrophoresis to separate a DNA fragment of about 7 kb, which was named fragment M13-T/R. A ligation reaction tube was provided with 100 ng of each of fragments BGHRAN-T/R and M13-T/R, 2 p2 of 10x ligation buffer, and 10 units of T4 DNA 1 0 ligase; and distilled water was added thereto to a total volume of 20 pe. The ligation was carried out at 16 0 C for 12 hours.
p 2 of the resulting ligation mixture was mixed with 200 p2 of E. coli JM105(ATCC 47016) competent cells, and to 15 the mixture were added 8 p£ of 0.2M IPTG solution, 100 p£ of :pre-cultured helper cells(E. coli JM105), 3 m2 of 2XYT upper agar(soft agar; 16 g Bactotryptone, 10 g yeast extract, 5 g NaC1, 5 g Bactoagar/2), and 25 p 2 of 4% X-gal. The mixture f* was spread on Min A plate(10.5 g KH 2
PO
4 1 g (NH 2
SO
4 0.5 g Na-citrate, 12 g Bactoagar, 1 ml 20% MgSO 4 0.5 ml 1% Vit. B, ml 50% glucose/2), and then cultured at 37 0 C for 12 hours.
The transparent plaques formed on the medium were picked, transferred to 2 ml of 2XYT liquid medium, and then cultured at 37 0 C for 5 hours. The culture was centrifuged to obtain a supernatant and 1/4 volume of polyethylene glycol, 2.5 M NaC1) was added to the supernatant. M13 phage was separated from the supernatant; 15 single-stranded DNA was extracted therefrom; and the nucleotide sequence of the 5'-end region of the BGH gene was confirmed(see Fig. 1).
Example 3: Construction of an Expression Vector Comprising trpA Transcription Terminator Gene (Step 1) 10 To amplify the trpA transcription terminator gene and insert it into the BGH expression vectors prepared in Example 2, the following primers were synthesized.
Primer PTRPATER: "15 CGCGCGTTTCGGT-3'.
Primer PTRPATER comprises a recognition site for SalI, and the 23rd-50th and the 51st-67th nucleotides thereof correspond to the trpA transcription terminator gene and the nucleotide sequence after the 3'-end of the BGH gene in the vector, respectively.
As shown below, plasmid PPBR3740 corresponds to a part of Amp" gene in plasmid ptrphs BGH 1-13 and comprises a recognition site for PvuI.
Plasmid PPBR3740: 5'-TGACAACGATCGGAGGACCGAAGGA-3'.
16 (Step 2) To a test tube were added 1 ng of plasmid ptrphs BGH 1-13 as a template, 2 pg of each of primers PTRPATER and PPBR3740 prepared in (Step 10 p2 of 10x polymerase reaction buffer(10 mM Tris-HC1, pH 8.3, 500 mM KC1, 15 mM MgCl 2 gelatin), 10 p. of 2 mM dNTP(each of dGTP, dATP, dCTP and dTTP is 2 mM), and 2.5 units of Taq DNA polymerase; and distilled water was added thereto to a final 10 volume of 100 The PCR was carried out by repeating times the cycle of: 95 0 C for 1 min., 55 0 C for 40 sec., and S. 72 0 C for 2 min.
The PCR product obtained above was subjected to 1% agarose gel electrophoresis to confirm that about 1.5 kb of 15 DNA was amplified. The DNA was purified by the same agarose gel electrophoresis as above and named fragment PBR.
S
08 (Step 3) s 2 pg of plasmid ptrphs BGH 1-13 was completely digested with Sall and PvuI in NEB buffer 3 and the resulting mixture was subjected to 0.7% agarose gel electrophoresis to isolate a fragment of about 1.5 kb, which was named fragment PBGH-
P/L.
2 pg of plasmid ptrphs BGHRAN #5 obtained in (Step 1) of Example 2 was completely digested with Sall and PvuI in NEB buffer 3 and the resulting mixture was subjected to 0.7% 17 agarose gel electrophoresis to isolate about 1.5 kb fragment, which was named fragment PBGHRAN-P/L. Further, 2 pg of fragment PBR obtained in (Step 2) was completely digested with SalI and PvuI in NEB buffer 3. The resulting DNA fragment was extracted with phenol/chloroform and then dissolved in 20 p of TE buffer. The DNA fragment was named fragment PBR-P/L.
A ligation reaction was carried out as follows by using the DNA fragments obtained above. Two reaction tubes A and B were provided with 100ng of each of fragments PBGH-P/L and PBGHRAN-P/L, respectively. To each of the tubes were added 100 ng of fragment PBR-P/L, 2 p of 10x ligation buffer and 10 units of T4 DNA ligase; and distilled water was added S. thereto to a total volume of 20 p 2 The ligation reaction 15 was carried out at 16 0 C for 12 hours.
E. coli W3110(ATCC 37339) cells were transformed with the ligation mixtures in accordance with the CaCl 2 method(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982) to obtain recombinant E.
coli transformants containing plasmid ptrp3H BGH 1-13(tube A) or plasmid ptrp3H BGHRAN(tube B)(see Fig. 2).
Example 4: Expression of BGH by Using Expression Vectors Comprising Modified BGH Gene and trpA Terminator E. coli transformants obtained in Example 3 were cultured with shaking in a liquid Luria medium(6% Bactoii~r~iF~V~7] 18 tryptone, 0.5% yeast extract, 1% NaC1) containing 50 pg/m£ of ampicillin at 37 0 C for 12 hours. 3 m2 of the culture was transferred into 300 m. of M9 medium(40 mM K 2
HPO
4 22 mM
KH
2
PO
4 8.3 mM NaCI, 18.7 mM NH 4 C1, 1% glucose, 0.1 mM MgSO 0.1 mM CaCI 2 0.4% casamino acid, 10 pg/m£ Vit. 40 pg/m2 ampicillin); and cultured with shaking at 37 0 C for 4 hours.
When the O.D. value of the culture at 650 nm reached about 0.3, indole acrylic acid(IAA) was added to the culture to a final concentration of 50 pg/m2. After 4 hours, the 10 O.D. of the resulting culture was determined and the culture was centrifuged with a centrifuge(Beckman J2-21, JA14 rotor) at 11,000 rpm for 25 min. to collect the E. coli cell precipitates. The cell precipitates were subjected to SDS-PAGE in accordance with Laemmli's method(Nature, 227, 15 680(1970)) to confirm the expression of BGH. The result is shown in Fig. 3.
As shown in Fig. 3, the amount of BGH produced by E.
coli cells transformed with plasmids ptrp3H BGH 1-13 or ptrp3H BGHRAN were 39.9% and 57.3%, respectively, on the basis of the total proteins produced in the cell. These amounts are 40-50% higher than that produced by E. coli transformed with plasmids ptrphs BGH 1-13 or ptrphs BGHRAN 26.3% and 39.9%, respectively(see Fig. 4).
E. coli W3110 transformed with plasmid ptrp3H BGHRAN was deposited on December 26, 1994 with the Korean Collection for Type Cultures(KCTC)(Address: GERI, KIST, P.O. Box 115, Yusong, Taejon, 305-600, the Republic of Korea) with the 19 accession number of KCTC 0143BP, under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims (5)

1. An expression vector for bovine growth hormone (BGH) comprising a promoter, a BGH gene and a terminator, said vector being characterised in that a part of a salmon growth hormone gene contained in a SacI/PvuI fragment of PCR-amplified plasmid ptrphs BGH 1-13 herein defined is present between said promoter and the 5'-end of the BGH gene; the terminator is trpA transcription terminator inserted no more than 50 nucleotides downstream of the stop codon of said BGH gene; and the nucleotide sequence at the 5'-end region of the BGH gene is modified for minimizing the formation of a secondary structure in mRNA and increasing the expression rate, without changing the amino acid sequence encoded therein.
2. The expression vector of claim 1, wherein the region of the modified bovine growth hormone gene includes the nucleotide sequence of: 5'-ATG GCT TTT CCG GCT ATG TCT CTA TCT GGC CTA TTC GCA AAT GCC GTT CTT CGA GCT CAG CAT CTT CAT CAG CTG GCT-3' wherein ATG is a translation initiation codon of the BGH gene. S 25 3. The expression vector of claim 1, wherein the trpA o* transcription terminator includes the nucleotide sequence of: ATGAGCGGGC TTTTTTTT-3' S S T
4. The expression vector of claim 3, which is plasmid trp3H BGHRAN (KCTC 0143BP) r7IifIFTPi7~[ ii[1Ti11flhiiiiiiii1Fi1~~~ hi 21 An E. coli cell transformed with the expression vector of any one of claims 1 to 4.
6. The E. coli cell of claim 5, which is E. coli W3110 transformed with plasmid ptrp3H BGHRAN(KCTC 0143BP).
7. A process for the mass production of bovine growth hormone, which comprises culturing the E. coli transformant of claim 5 and separating bovine growth hormone from the 10 culture. DATED this 27th day of December 1995 L G CHEMICAL LTD. By their Patent Attorneys CULLEN CO. *oo
AU40768/95A 1994-12-30 1995-12-28 Process for the mass production of bovine growth hormone Ceased AU713218B2 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1988005078A1 (en) * 1986-12-31 1988-07-14 Lucky, Ltd. Method for the production of bovine growth hormone using a synthetic gene
EP0496327A1 (en) * 1991-01-22 1992-07-29 Grünenthal GmbH Polypeptides with prourokinase activity, plasmids coding therefor and process for their preparation and use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005078A1 (en) * 1986-12-31 1988-07-14 Lucky, Ltd. Method for the production of bovine growth hormone using a synthetic gene
EP0496327A1 (en) * 1991-01-22 1992-07-29 Grünenthal GmbH Polypeptides with prourokinase activity, plasmids coding therefor and process for their preparation and use

Non-Patent Citations (1)

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Title
J. BIOCHEM, 101, PP 525-534 (1987) *

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