JPH08228787A - Method of mass-producing cattle growth hormone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new expression vector capable of producing a bovine growth hormone in a high yield, by including a modified bovine growth hormone gene in which a base sequence at its 5'-end region is changed without changing an amino acid sequence at the end region.

SOLUTION: The new modified bovine growth hormone gene contains the 5'-end part of a base sequence of formula I (ATG indicates a translation initiation codon of a bovine growth hormone gene) obtained by changing only the base sequence of the 5'-end part without changing an amino acid sequence encoded in the end part, and the new bovine growth hormone expression vector comprises a trp transcription terminator having a base sequence of formula II bound to the 3'-end of the gene. The bovine growth hormone can be mass-produced by transferring the expression vector into Escherichia coli to give a transformed transformant, culturing the transformant to express the bovine growth hormone gene.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the mutation of a part of the 5′-terminal gene of bovine growth hormone without changing the amino acid sequence encoded by the terminal, and the 3′-terminal of bovine growth hormone. The present invention relates to a method for producing only bovine growth hormone in a large amount by using an expression vector obtained by ligating a trp A transcription terminator gene after the above.

More specifically, Korean Patent Application No. 86-
Using the nucleotide sequence of the bovine growth hormone gene disclosed in No. 11712, the amino acid sequence encoded by the 5'-terminal portion of the gene is not changed, but only the nucleotide sequence of the 5'-terminal portion is randomly changed. Primer
, A bovine growth hormone gene was amplified in large amounts using the same, and an expression vector containing a bovine growth hormone gene obtained by ligating a trpA transcription terminator to the 3'-end of the gene was used. A method for mass-producing growth hormone protein is provided.

[0003]

2. Description of the Related Art Animal growth promoters used so far are steroid-based products (eg, Estradiol-Compudose, manufactured by Eli Lilly, Est.
radiol Benzoate-Synovax, Syntax Agribusiness Inc.
Zeramol-Ralgro, International Minerals and C
hemicals), but these are fat-soluble (fat-solub
Since it is a le), it remains in the body of animals, and it has become clear that ingestion of this will adversely affect the human body, and its use is currently banned in developed countries including the United States.

On the other hand, growth hormone produced and secreted in the pituitary gland of animals is insoluble and species-specific (Species
-Specific) was shown, and it was found to have the effects of promoting animal growth, increasing milk secretion of dairy cows, and improving feed efficiency.
(Reference: Bauman, DE et al., Journal of Animal Sci
ence, 60 , 583 (1985); Hart, IC, et al., Journal of Endo
crinology, 105 , 189 (1985); Newswatch, June 17,1985; Wa
ll Street Journal, July 22, 1986). Such growth hormones have been directly extracted from the pituitary gland of animals since 1940,
Although it was purified and used for research purposes, the amount that could be produced by this method was extremely limited and could not be put to practical use for the livestock industry. However, recent developments in genetic engineering technology have enabled large-scale production of such animal growth hormone using Escherichia coli.

Seeburg et al. Isolated a cDNA encoding bovine growth hormone from bovine pituitary gland to elucidate the nucleotide sequence and amino acid sequence, and identified this gene.
It was cloned into an E. coli expression vector using the trp promoter and expressed (Seeburg, PH, et al., DNA , 2 , 37
(1983)). After that, many research groups tried to increase the expression rate of bovine growth hormone.

[0006] George et al. Revealed that the expression rate changes depending on the sequence and length of the base between the ribosome binding site of the bovine growth hormone gene and the translation initiation codon ATG. ,
By controlling the base at this site, bovine growth hormone was expressed at 15% of the total E. coli protein (George HJ, e
al., DNA, 4 , 273 (1985)). Olsen et al. Used an Escherichia coli expression vector containing a large proportion of AT base pairs in the nucleotide sequence between the ribosome binding site of the bovine growth hormone gene and the translation initiation codon ATG and an E. coli expression vector containing the trp promoter. As a result of expressing the growth hormone gene, it was reported that bovine growth hormone was produced at 15% to 20% of total E. coli protein (Olsen, MK, et al., J. Bio.
technol., 9 , 179 (1989)). As a result of random mutation of the plasmid into which the bovine growth hormone gene was cloned, Watson et al. Found that one base in the N-terminal 4 amino acid-encoding nucleotide sequence of bovine growth hormone was Substituted mutants were obtained and reported that 20% of total E. coli protein expressed bovine growth hormone using this mutant (Watson, et al., Gene , 86 , 137).
(1990)). The present inventor also modified the bovine growth hormone gene with a codon compatible with Escherichia coli, cloned this into an E. coli expression vector having a salmon growth hormone gene under the control of the trp promoter, and defined the ribosome binding site and the initiation codon ATG. A synthetic linker was ligated between the salmon growth hormone gene and the bovine growth hormone gene so that an mRNA secondary structure would not be generated between them, to prepare an expression vector for bovine growth hormone, which was then expressed in E. coli. Reported that it was able to produce bovine growth hormone with 27% of total E. coli protein (Korea Patent Publication No. 92)
-3665).

On the other hand, the trpA transcription terminator has been reported to be a very efficient rho-independent terminator (Christie, GE, et al., Pr.
oc.Natl.Acad.Sci.USA, 78 , 4180 (1981)), a trpA transcription terminator was inserted at the 3'-end of the gene to be expressed to block an unnecessary transcription process, and thus the It has been reported that only the desired protein can be mass-produced while suppressing the required protein production (Gentz, R.,
et al., Proc. Natl. Acad. Sci. USA, 78 , 4936 (1981)).
In fact, Matsuki et al.
(n) Expression of a vector containing a gene modified by the trp promoter and the trpA transcription terminator in Escherichia coli resulted in the expression of mouse calmodulin in 30% of the total E. coli protein (Matsuki, S., et
al., Biotech.Appl.Biochem., 12 , 284 (1990)), and Sa
to et al. reported that when the interleukin-2 gene was expressed in Escherichia coli in the presence of the trp promoter, the expression rate was increased by about 5 times by inserting the trpA transcription terminator (Sato, et al. al., J. Biochem.,
101 , 525 (1987)).

[0008]

However, in the case of producing bovine growth hormone by the above method, the ratio of E. coli total protein is 30%.
There is a limit that the above yield cannot be obtained. The present inventor has made diligent efforts to produce an expression vector capable of further increasing the expression rate of bovine growth hormone, and as a result, 5'of bovine growth hormone gene has been obtained.
-Synthesizing a bovine growth hormone gene using a random primer capable of randomly changing only the base sequence of the portion without changing the amino acid sequence encoded by the terminal portion, and then the bovine growth hormone gene and 3 of the gene are synthesized. When an expression vector containing a trpA transcription terminator inserted after the'-end was expressed in Escherichia coli, the total ratio of E. coli was 50%.
It has been discovered that bovine growth hormone can be expressed in large amounts with a high yield of over%.

Therefore, an object of the present invention is to change the gene base sequence of the 5'-end of the bovine growth hormone gene to mRN.
It contains a gene that minimizes the formation of secondary structure of A and inserts a trpA transcription terminator after the 3'-end, and has a total protein ratio of 50 in E. coli.
It is intended to provide an expression vector capable of inducing bovine growth hormone expression of 10% or more and a method for producing the same, and to provide a large amount of bovine growth hormone at a low cost accordingly.

According to the present invention, the 5'-terminal nucleotide sequence of the bovine growth hormone gene is changed without changing the sequence of the amino acid encoded at this end, and further the 3'- of the gene. Trp linked to the end
The present invention provides an expression vector containing an A transcription terminator gene, Escherichia coli transformed with the vector, and a method for producing bovine growth hormone using the same.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.
The expression vector of the present invention is 5′-of bovine growth hormone gene.
A bovine growth hormone gene whose nucleotide sequence is modified to enhance the expression rate of the gene by minimizing the formation of secondary structure of bovine growth hormone mRNA;
Preferably, it includes a 5'-terminal portion having the following nucleotide sequence (SEQ ID NO: 1): 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 '

The expression vector of the present invention also contains a trpA transcription terminator linked to the 3'-end of the bovine growth hormone gene, and the trpA transcription terminator preferably has the following nucleotide sequence (SEQ ID NO: 2). 5'-AGCCCGCCTA ATGAGCGGGC TTTTTTTT- 3 '

The expression vector of the present invention as described above can be produced by a known gene recombination or synthesis method, for example, the following method. First, based on the nucleotide sequence information encoding bovine growth hormone (Korean Patent Publication No. 92-3665), a polymerase chain reaction method (polymerase chain reaction) performed to change the 5'-terminal nucleotide sequence of the bovine growth hormone gene. PC
R) is used to synthesize a random primer. This random primer is designed so that the bovine growth hormone gene contains a recognition site for the restriction enzyme SacI, and is designed so that only the base sequence changes randomly without changing the amino acid sequence. Also, a recombinant vector ptrphs BGH 1-13 (Korean patent publication No. 92-3665: ATCC 68975 (1992.) in which the bovine growth hormone gene was cloned.
5.6))), a primer designed to contain a unique restriction enzyme PvuI recognition site was synthesized, and then the polymerase chain reaction was performed using this primer and a random primer to prepare 5'of the bovine growth hormone gene. -A gene fragment obtained by amplifying a mutant gene with a changed base sequence at the terminal and cutting the amplified gene with a restriction enzyme and the ptrphs BGH 1-13 recombinant vector with the restriction enzymes PvuI and SacI After ligation with the resulting fragment, E. coli is transformed to obtain the ptrphs BGHRAN plasmid.

For the purpose of inserting the trpA transcription terminator into the bovine growth hormone expression vector, trpA
After synthesizing the primer so that it contains the transcription terminator gene and the restriction enzyme SalI recognition site, and also synthesizing the primer so that the primer contains the only restriction enzyme PvuI recognition site of the ptrphs BGHRAN recombinant vector, these primers are used. After carrying out the polymerase chain reaction to amplify the gene, the gene fragment obtained by cleaving the amplified gene with a restriction enzyme and ptrphs BG
After ligating the gene fragment obtained by cutting the HRAN plasmid with the restriction enzymes PvuI and SalI, E. coli was transformed with the ligation mixture to obtain the ptrp3H BGHRAN plasmid.

Escherichia coli suitable for expression is transformed using such an expression vector of the present invention to prepare an E. coli transformant, and the E. coli transformant is cultivated in an appropriate medium. Rate of bovine growth hormone is separated and purified to produce.

The present invention will be described in more detail based on the following examples, but the following examples illustrate the present invention and are not intended to limit the same. Example 1: Amplification of bovine growth hormone gene having modified 5'-terminal and production of modified bovine growth hormone expression vector (Step 1) Cloned nucleotide sequence of bovine growth hormone gene (Korea Patent Publication No. 92-3665), the following random primers were synthesized. Primer PBGHRAN (5'-TGCTGAGCTC GNAGNACNGC RTTNGCRA
AN AGNCCNGANA GNGACATNGC NGGRAANGCC ATTTATAATT CCT
CCA-3 ') (SEQ ID NO: 3) has a restriction enzyme SacI recognition site, and has only the base sequence without changing the 16 amino acid sequence encoded by the 5'-terminal portion of the bovine growth hormone gene. It has been changed. In the above sequence, N is A,
T, G or C, and R is A or G. Primer PPBR3720 (5'-TCCTTCGGTC CTCCGATCGT TGTC
A-3 ') (SEQ ID NO: 4) is located in the Amp ^r gene of bovine growth hormone expression vector ptrphs BGH 1-13 and contains a restriction enzyme PvuI recognition site.

(Step 2) Bovine growth hormone expression vector ptrphs BGH 1-13 (Korean Patent Publication No. 9) in a reaction tube
No. 2-3366, ATCC 68975) 1 ng and 2 μg of primer PBGHRAN and 3 μg of PPBR 3720 prepared in step 1 were placed in a template and then 10 μl of 10 × polymerase reaction buffer solution, 10 μl.
In the case of μl, dNTP (containing 2 mM each of dGTP, dCTP, dATP and dTTP) and 2.5 units of Taq polymerase were added, and the total volume was adjusted to 100 μl with distilled water.
1 minute (denaturation) at 5 ℃, 40 seconds (annealin at 55 ℃)
g), the polymerase chain reaction was performed 25 times at 72 ° C. for 2 minutes (polymerization). PCR product obtained here is 5
About 9 as a result of separation using a% polyacrylamide gel
It was confirmed that a 90 base pair DNA was amplified, and this DNA was separated and purified using the same polyacrylamide gel. Hereinafter, this fragment is referred to as BGHRAN.

(Step 3) Plasmid ptrphs BGH 1-
NEB (New England Biolab.) Buffer Solution 1 (10 mM Bis Trispropane-HCl, 10 mM Mg)
Cl _2, 1 mM DTT, was completely cleaved with the restriction enzymes SacI at pH 7.0) medium, followed by NEB buffer solution 3
(100 mM NaCl, 50 mM Tris-HCl, 10 mM
Restriction enzyme P in MgCl ₂ , 1 mM DTT, pH 7.9)
Completely cleaved with vuI and about 2 kb fragment was separated using 0.7% agarose gel. This fragment will be referred to as fragment PBG below.
It is called HT / P. On the other hand, the fragment BGHR obtained in step 2
The AN was completely cleaved with the restriction enzyme SacI in the NEB buffer solution 1, and then the restriction enzyme P was further added in the NEB buffer solution 3.
After completely cleaving with vuI, it was extracted with phenol / chloroform and dissolved in 20 μl of TE solution. The fragment thus obtained is called fragment BGHRAN-T / P.

The conjugation reaction was carried out as follows using the fragments obtained above. In a reaction tube, the fragment BGHRAN-T /
P100 ng, fragment PBGH-T / P100 ng, 2 μl of 10 × conjugation buffer solution and 10 units of T4 DNA ligase were added and distilled water was added so that the total volume became 20 μl.
The reaction was carried out at 16 ° C for 12 hours. Escherichia coli W3110 (ATCC 37339) was transformed with this ligation product to obtain plasmid ptrphs BGH containing the fragment BGHRAN.
A RAN was obtained (Figure 2).

Example 2: Induction of expression of bovine growth hormone gene having a modified 5'-terminal nucleotide sequence and confirmation of its nucleotide sequence (step 1) Recombinant E. coli colony 10 obtained in Example 1
After culturing 0 or more cells in a liquid Luria medium (6% bactotripton, 0.5% yeast extract, 1% sodium chloride) containing 50 μg / ml of ampicillin for 12 hours, this culture solution was used. 3 ml to 300 ml of M9 medium (40 mM K
₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.5 mM NaCl,
18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM M
gSO ₄ , 0.1 mM CaCl ₂ , 0.4% casamino acid, 10μ
g / ml Vit.B1, 40 μg / ml ampicillin) and cultivated with shaking at 37 ° C. for about 4 hours. When the absorbance of the bacteria at a wavelength of 650 nm reached about 0.3, indole acrylic acid, IAA) final concentration 50 μg
/ Ml was added. Approximately 4 hours after adding IAA, measure the absorbance of the cell culture solution, and then centrifuge (Bec
25 km at 11,000 rpm using kman J2-21, JA14 rotor)
Bacterial cell precipitate was collected by centrifugation for minutes and the cell precipitate was collected by the Laemmli method (Laemmli, Nature, 227 , 680 (1970)).
The expression level of bovine growth hormone was measured by electrophoresis using a 15% polyacrylamide gel in the presence of SDS. As a result, the plasmid ptrphs BGH 1-1
A clone in which the expression level of bovine growth hormone was increased by 40% as compared with the control E. coli containing 3 was selected, and the plasmid contained in this was designated as ptrphs BGHRAN # 5 (FIG. 3).

(Step 2) ptrphs selected in Step 1
An experiment by the method of Sangar et al. Was performed as follows in order to confirm the nucleotide sequence of the BGHRAN # 5 clone. First, the plasmid ptrphs BGHRAN # 5
2 μg was completely digested with the restriction enzyme SacI in NEB buffer solution 1, then completely digested with the restriction enzyme EcoRI in NEB buffer solution 3, and a DNA fragment of about 187 base pairs was digested with 7% acrylamide gel. Was separated and purified.
Hereinafter, this DNA fragment is referred to as fragment BGHRAN-T / R. On the other hand, 2 μg of the plasmid M13mp18 was completely cleaved with NEB buffer solution 1 using the restriction enzyme SacI.
Completely cleaved with EB buffer solution 3 using the restriction enzyme EcoRI. Thereafter, the reaction mixture was subjected to 0.7% agarose gel to separate a DNA fragment of about 7 kb. Hereinafter, this DNA fragment is referred to as fragment M13-T / R. The fragment BGHRA
NT-R100ng, fragment M13-T / R100ng, 2
Add 10 μl of 10x conjugation buffer solution and 10 units of 4 DNA ligase to the conjugation reaction vessel and add 20 μl total volume with distilled water.
Then, the mixture was reacted at 16 ° C. for 12 hours. 10 μl of this ligation product was used for Escherichia coli JM105 (ATCC 47016)
After mixing with 200 μl of competent cells,
8 μl of 0.2 M IPTG solution, 100 μl of pre-cultured helper cells (E. coli JM105)
l, 3 ml of 2XYT top agar (soft agar: 16 g bactotripton, 10 g yeast extract, 5 g NaCl, 5 g bacto agar / l) and 25 μl of 4% X-gal solution were added.
This mixture was mixed with Min A plate (10.5 g KH ₂ P).
O ₄ , 1 g (NH ₄ ) ₂ SO ₄ , 0.5 g sodium citrate,
12g Bact agar, 1ml 20% MgSO ₄ , 0.5ml 1% V
ItB, 10 ml of 50% glucose / l) and then incubated at 37 ° C. for 12 hours, and the resulting clear plaques were selected and transferred to 2 ml of 2XYT liquid medium and incubated at 37 ° C. for 5 hours. Then, 1/4 volume of PEG / NaCl (20% polyethylene gl) was added to the supernatant obtained by centrifugation.
ycol, 2.5M NaCl, and then M13 phage (p
hage) was separated, single-stranded DNA was extracted, and the sequence of the 5'-terminal base of the bovine growth hormone gene was confirmed (Fig. 1).

Example 3: Preparation of expression vector having trpA transcription terminator gene (step 1) The following primers were synthesized to insert the trpA terminator gene into the bovine growth hormone expression vector prepared in Example 2 above. . Primer PTRP
ATER (5'-GCTTTGTCGA CTAATTAAAG CCCGCCTAAT GAG
CGGGCTT TTTTTTGCCT CGCGCGTTTC GGT-3 ') (SEQ ID NO:
5) contains a recognition site for the restriction enzyme SacI,
The 0th base portion shows the trpA transcription terminator gene, and the 51st to 67th base portions show the base sequence from the 3'end of the bovine growth hormone gene in the expression vector. Primer PPBR3740 (5'-TG
ACAACGAT CGGAGGACCG AAGGA-3 ') (SEQ ID NO: 6) is located in the Amp ^r gene of bovine growth hormone expression vector ptrphs BGH 1-13 and contains a restriction enzyme Pvu I recognition site.

(Step 2) 1 ng of bovine growth hormone expression vector ptrphs BGH 1-13 1 ng was placed in a reaction tube together with 2 μg of the primer PTRPATER prepared in Step 1 and 37402 μg of the primer PPBR, and then 10
μl of 10 × polymerase reaction buffer solution, 10 μl of dNTP (containing 2 mM each of dGTP, dATP, dCTP and dTTP) and 2.5 units Taq polymerase to make a total volume of 100 μl, and then 1 at 95 ° C. Polymerization chain reaction was performed 25 times under conditions of 55 minutes, 55 ° C. for 40 seconds, and 72 ° C. for 2 minutes. The generated PCR product was separated using 1% agarose gel, and as a result, D of about 1.5 kb base pair was obtained.
It was confirmed that NA was amplified, and this DNA was separated and purified using the same agarose gel. Hereinafter, this DNA fragment is referred to as a fragment PBR.

(Step 3) Plasmid ptrphs BGH 1-
13 (ATCC 68975) 2 μg in NEB buffer solution 3
Digested with restriction enzymes SacI and PvuI in
A DNA fragment of about 1.5 kb was separated and purified using a 7% agarose gel. Hereinafter, this DNA fragment is referred to as fragment PBGH-P.
/ L. The plasmid obtained in step 1 of Example 2 above
ptrphsBGHRAN # 5 2 μg in NEB buffer solution 3
Completely cleaved with restriction enzymes SalI and PvuI in
A DNA fragment of about 1.5 kb was separated using a 7% agarose gel. Hereinafter, this DNA fragment is referred to as the fragment PBGHRAN-
It is called P / L. On the other hand, the fragment PBR obtained in step 2 was completely cleaved in the NEB buffer solution 3 with the restriction enzymes SalI and PvuI, and the resulting fragment was extracted with phenol / chloroform and dissolved in 20 μl of TE solution. Hereinafter this fragment is referred to as PBR-P / L.

The ligation reaction was carried out as follows using the fragment PBR-P / L. Fragment PBGH-P in conjugation reaction tube A
/ L 100 ng was added to the conjugation reaction tube B with the fragment PBGHRAN.
-P 100 L in each tube after adding 100 ng of P / L
ng fragment PBR-P / L, 2 μl of 10 × conjugation reaction solution, and 10 units of T4 DNA ligase were added, and the total volume was adjusted to 20 μl with distilled water, followed by reaction at 16 ° C. for 12 hours. Escherichia coli W3110 (ATCC 37339) was transformed with the ligation reaction product according to the Cell method, and as a result, p containing the trpA transcription terminator from the reaction tube A was obtained.
trp3H BGH1-13 and trpA transcription terminator-containing ptrp 3HBGHRAN were obtained from reaction tube B (see FIG. 2).

Example 4: Induction of bovine growth hormone expression using bovine growth hormone expression vector containing trpA transcription terminator Plasmid ptrphs BGH 1-13, ptrphs BGH
RAN # 5, ptrp3H BGH 1-13 and ptrp
Recombinant E. coli cells containing 3H BGHRAN were each added to liquid Luria (Lu) containing 50 μg / ml ampicillin.
ria) medium (6% bactotripton, 0.5% yeast extract, 1% sodium chloride), after culturing with shaking for 12 hours,
3 ml of each culture solution was added to 300 ml of M9 medium (40 mM).
K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaC
1, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM
MgSO ₄ , 0.1 mM CaCl ₂ , 0.4% casamino acid,
10 μg / Vit.B1, 40 μg / ml ampicillin) and cultured with shaking at 37 ° C. for about 4 hours. When the absorbance of the bacteria at a wavelength of 650 nm was about 0.3, indole acrylic acid (IAA) was added. ) To a final concentration of 50
It was added so as to be μg / ml. About 4 after adding IAA
Centrifuge after measuring the absorbance of the cell culture medium after a period of time
(Beckman J2-21, JA14 rotor) was centrifuged at 11,000 rpm for 25 minutes to collect bacterial cell precipitates, which were collected by the Laemmli method (Laemmli, Nature, 227 , 680 (19)).
FIG. 3 shows the results of confirming the expression of bovine growth hormone by electrophoresis on a 15% polyacrylamide gel in the presence of SDS according to 70)).

As shown in FIG. 3, the plasmid ptrp3
Bovine growth hormone expressed in E. coli transformed with HBGH 1-13 and plasmid ptrp3
Bovine growth hormone expressed using Escherichia coli transformed with HBGHRAN was 3% of E. coli total protein, respectively.
The expression rates were 9.9% and 57.3%. This incidence is
The expression rate of bovine growth hormone was 26.3% when Escherichia coli transformed with the plasmid ptrp3H BGHRAN 1-13 was used, or the plasmid ptrphs BGH
The expression rate of bovine growth hormone when using E. coli transformed with RAN # 5 was 40 to 5 compared with the expression rate of 39.9%.
It was found to be 0% higher (see Figure 4). Plasmid ptrp
E. coli W3 transformed by 3H BGHRAN
110 was deposited on December 26, 1994 at the Genetic Engineering Research Institute Gene Bank (KCTC) attached to the Korea Institute of Science and Technology with the deposit number KCTC 0143BP.

[0028]

Industrial Applicability As described above, the present invention is produced by minimizing the formation of mRNA secondary structure by nucleotide sequence conversion of the 5'-terminal portion of the bovine growth hormone gene and inserting a trpA transcription terminator at the 3'-terminal of the gene. The expression vector can be used to produce bovine growth hormone in a high yield of 57.3% of total E. coli protein, which provides a means for economically mass producing bovine growth hormone.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 78 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence ATG GCT TTT CCG GCT ATG TCT CTA TCT GGC CTA 33 TTC GCA AAT GCC GTT CTT CGA GCT CAG CAT CTT 66 CAT CAG CTG GCT 78

SEQ ID NO: 2 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence AGCCCGCCTA ATGAGCGGGC TTTTTTTT 28

SEQ ID NO: 3 Sequence length: 76 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Primer DNA sequence TGCTGAGCTC GNAGNACNGC RTTNGCRAAN AGNCCNGANA GNGACATNGC 50 NGGRAANGCC ATTTATAATT CCTCCA 76

SEQ ID NO: 4 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Primer DNA Sequence TCCTTCGGTC CTCCGATCGT TGTCA 25

SEQ ID NO: 5 Sequence length: 63 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Primer DNA sequence GCTTTGTCGA CTAATTAAAG CCCGCCTAAT GAGCGGGCTT TTTTTTGCCT 50 CGCGCGTTTC GGT 63

SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Primer DNA sequence TGACAACGAT CGGAGGACCG AAGGA 25

SEQ ID NO: 7 Sequence length: 78 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence ATG GCT TTT CCG GCT ATG TCT CTA TCT GGT CTA 33 TTC GCT AAC GCT GTT CTT CGA GCT CAG CAT CTT 66 CAT CAG CTG GCT 78

[Brief description of drawings]

FIG. 1: Bovine growth hormone gene (ptrphs BG)
H 1-13) 5'-terminal part and this mutant (ptrphs BG
HRAN # 5) base sequence comparison,

FIG. 2A is a diagram showing the production process of a plasmid ptrphs BGHRAN containing a modified bovine growth hormone gene,

FIG. 2B: Plasmid ptrp3H BGH 1-1 containing the bovine growth hormone gene or its variant and the trpA transcription terminator inserted at its 3′-end, respectively.
3 is a diagram showing the production process of 3 and ptrp3H BGHRAN,

FIG. 3: Plasmid ptrphs BGH 1-1
3, ptrphs BGHRAN # 5, ptrp3H BGH
1 shows the results of SDS-polyacrylamide gel electrophoresis (PAGE) using E. coli cell pellets transformed with 1-13 or ptrp3H BGHRAN,

FIG. 4: Plasmid ptrphs BGH 1-1
3, ptrphs BGHRAN # 5, ptrp3H BGH
1 shows the results of measuring the amount of bovine growth hormone expressed in E. coli cells transformed with 1-13 and ptrp3H BGHRAN with a densitometer.

FIG. 6 (continued)

FIG. 7 FIG. 4 (continued)

FIG. 8 FIG. 4 (continued)

Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12P 21/02 C12R 1:19)

Claims (7)

[Claims] 1. A modified bovine growth hormone gene obtained by changing only the base sequence of the 5′-terminal portion without changing the amino acid sequence encoded by the 5′-terminal portion, and linked to the 3′-terminal of the gene. A bovine growth hormone expression vector, characterized in that it comprises a defined trpA transcription terminator. 2. The modified bovine growth hormone gene 5′-
The expression vector according to claim 1, wherein the terminal portion has the following nucleotide sequence (SEQ ID NO: 1): 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 ', where ATG refers to the translation initiation codon of the bovine growth hormone gene. 3. The expression vector according to claim 1, wherein the trpA transcription terminator has the following nucleotide sequence (SEQ ID NO: 2): 5′-AGCCCGCCTA ATGAGCGGGC TTTTTTTT-3 ′. 4. The expression vector ptrp3H according to claim 3.
BGHRAN (KCTC 0143BP). 5. Escherichia coli transformed with the expression vector according to any one of claims 1 to 4. 6. The E. coli W3110 according to claim 5, which has been transformed with the vector ptrp3H BGHRAN.
(KCTC 0143BP). 7. A method for producing bovine growth hormone, which comprises isolating bovine growth hormone after culturing the Escherichia coli according to claim 5 or 6.