BG100264A - Method for the preparation of bovine growth hormone - Google Patents

Abstract

The invention relates to a recipient vector comprising a bovine growth hormone (BGH) gene in which the nucleotide sequence at its 5'-terminal region is without alteration of the amino acid sequence encoded therein. A trpA transcriptional termination gene introduced after the 3'-terminus of the modified BGH gene for transforming E. coli cell for BGH mass production was used.

Description

The present invention relates to a process for the mass production of bovine growth hormone (BHP) and in particular to: a receiving vector comprising a modified BHP gene in which the nucleotide sequence in its 5'-terminal field is modified without alteration of the amino acid a sequence encoded therein, as the trpA copy-finishing gene is inserted after the 3'-end of the modified BHP gene; E. coli cell transformed with the receiving vector; and a process for mass production of VHP that involves culturing an E. coli transformant and separating VHP from the culture.

FIELD OF THE INVENTION

Steroidal animal growth promoters such as Eli Lilly, Syntax Agribusiness Inc. and International Minerals and Chemicals have been used in animal husbandry to increase nutrition and weight gain .

The use of such steroidal growth promoters is restricted, ie. they are fat-soluble and therefore have a long period of presence in animal tissues, which can be harmful to human consumption.

In contrast, animal growth hormones, which are produced and secreted by the pituitary glands of the animal, are not fat-soluble and show species specificity; they have been shown to increase the growth and yield of livestock milk and to increase feeding efficiency (Bauman, D. E., et al., J. of Animal Sciences, 60, 583 (1985); Hart, IC et. al., J. of Endocrinology, 105, 189 (1985); Newswatch, Jyne 17, 1985; and Wall Street Jounal, July 22, 1986).

Om 1940 -me naturally produced animal growth hormones, separated and purified from pituitary glands of animals, are used to stimulate animal growth, but their use in animal husbandry is limited due to the limited amount of hormones produced. Recent advances in genetic technology, however, make it possible to mass produce animal growth hormones by using microorganisms, such as E. coli.

Seiberg isolated cDNA encoded by BMP from the pituitary gland; determines the nucleotide sequence of cDNA and the amino acid sequence of BHR; it clones the E. coli cDNA into a receiving vector including a trpA stimulator, and receives the HPP into E. coli that is transformed with the vector (DNA, 2, 37 (1983)).

Subsequently, many attempts have been made to increase the resulting amount of VHP in E. coli.

For example, George finds that the amount obtained depends on the nucleotide sequence and the length of the region between

ribosomal localization and translation start codon, ATG; produces BMP in an amount equivalent to 15% of the total amount of proteins produced in E. coli (DNA, 4, 273 (1985)).

Olson obtains BMP in an amount of 10 to 15% based on the total amount of proteins produced in an E.coli cell by culturing the cell modified with a receiving vector comprising a modified BHP gene in which the region between the ribosomal localization and the translation start codon, ATG is an AT-rich, trp stimulating agent (J. Biotechnol., 9,179 (1989)).

Watson obtains HRP in 20% amounts based on the total amount of protein produced in an Exoli cell by culturing a cell modified with a protoplasm comprising a modified BHP gene, where one of the nucleotide-encoded four amino acids in the N-terminal region by HPLC mutation (Gene, 86, 137 (1990)).

It has also been reported that a modified BHP gene has been prepared using codons preferred by E. coli; a receiving vector for BHP has been constructed by introducing the BHP gene into an E.coli vector comprising a salmon growth hormone gene under the control of a trp stimulator and ligation of a synthetic link between the salmon growth hormone gene and the BHP gene; HPLC is produced in an amount of 27% of the total amount of proteins obtained by culturing an E.coli cell transformed with the vector (Korean patent N 92-3665).

The maximum amount of BMP that can be produced using processes of the prior art is limited to 30% of the total amount of proteins in E. coli.

On the other hand, the trpA copy end gene was found to be a very effective po-independent end gene (Christie, G. E. et al., Proc. Natl. Acad. Sci. USA, 78,4180 (1981)), and therefore, the required protein can be mass produced while limiting the production of unnecessary proteins derived from the protoplasm by introducing a trpA copy-finishing gene after the 3'-end of the gene, encoding the required protein (Gentz, R., et al., Proc. Nat. Acad. Sci. USA, 78, 4936 (1981).

Matsuki receives rat calmodulin in 30% of the total protein in E. coli by culturing a cell transformed with a protoplasm comprising a trp stimulator, a camodulin gene, and a trpA copying end gene (Biotech. Appl. Biochem., 12, 284 (1990) )).

Camo reports that when an interleukin-2 gene is obtained by using a protoplasm comprising an interleukin-2 gene under the influence of a trp stimulator and a trpA copy-finishing gene introduced after the 3'-end of the intelleukin-2 gene, the amount of the latter is about 5 times greater than that produced by a control group 6 which the trpA termination gene was not introduced (J. Biochem. 101, 525 (1987)).

-3 Despite these developments, there is still a need to develop a process for mass production of HCVs in terms of commercial and economic feasibility.

It is an object of the present invention to provide a receiving vector suitable for the mass production of bovine growth hormone; provide a host cell transformed with the receiving vector; to provide a process for the mass production of bovine growth hormone, which involves the cultivation of the transformant.

In accordance with the present invention, there is provided an improved process for the mass production of bovine growth hormone by constructing a receiving vector comprising a modified bovine growth hormone gene in which the nucleotide sequence is in 5'-kr. its field was modified without alteration of the amino acid sequence encoded therein, and the trpA copy-finishing gene was inserted after the 3 'end of the modified bovine growth hormone gene; the production of bovine growth hormone is in amounts greater than 50% relative to the total amount of protein obtained by culturing an E. coli cell converted to said vector.

Description of the drawings

The foregoing and other objects and features of the present invention are depicted by way of description in conjunction with the figures as follows:

FIG. 1 compares the nucleotide sequence 6 5 'cr. the field of the bovine growth hormone gene (ptrphs BHP 1-13) and in its variant (ptrphs BGHRAN # 5);

FIG. 2A is a schematic diagram for constructing a protoplasmic ptrphs BGHRAN comprising a modified BHP gene;

FIG. 2B is a schematic diagram for constructing protoplasmic ptrp3H BMP 1-13 and ptrp3H BGHRAN, including the BHP gene or variant thereof, together with a trpA copy finishing gene ligated into 3 'cr. respectively;

FIG. 3 presents the result of SDS-polyacrylamide gel electrophoresis (PAGE) using an E. coli cell pellet transformed with protoplasmic ptrhs BMP 1-13, ptrphs BGHRAN # 5, ptrp3H or ptrp3H BGHRAN;

FIG. 4 presents the results of a densimeter determination of the amount of bovine growth hormone produced in E, coli cells transformed with protoplasmic ptrphs BHP 1-13, ptrphs BGHRAN # 5, ptrp3H BHP 1-13 and ptrp3H BGHRAN respectively.

Description of the invention

-4According to the objectives of the present invention, a receiving vector for mass production of VHP is provided, which includes a modified VHP gene and a trpA copy finishing gene introduced after 3'-kr. of the modified VHR gene.

The modified BHP gene of the present invention has a modified nucleotide sequence in its 5'-kr. field to minimize secondary structures in the mRNA and increase the yield, while encoding the unchanged amino acid sequence of normal bovine growth hormone. The modified BHP gene of the present invention is preferably present in 5'-kr. its nucleotide sequence is as follows:

C 5 '- ATG GCT TTT CCG GOT ATG TCT CTA TCT GGC

CTA TTC GCA AAT GCC GTT CTT CGA GCT CAG CAT CTT CAT CAG CTG GCT-3 ', where ATG is a translation start codon of the BHP gene.

The receiving vector of the present invention also includes a trpA copy-finishing gene after 3 '-cr. of the modified BHP gene, which preferably has a nucleotide sequence as follows:

5 '-AGCCCGCCTA ATGAGCGGGC TTTTTTT-3'

The receiving vector of the present invention may be prepared in accordance with any conventional genetic recombination or genetic synthesis method, for example, by the procedure described below.

First, random primary chains are synthesized based on information from the nucleotide sequence of the BHP gene (see Korean Patent No. 92-3665). These random chains are intended to introduce into the BHR the ability to recognize the Saci restriction enzyme and to randomly alter the nucleotide sequence encoding BHR without altering the encoded amino acid sequence.

A primary strand has also been prepared that corresponds to a portion of the Atr gene in recombinant ptrphs carrier BMP 1-13, including the BHP gene (see Korean Patent No. 92-3665, ATCC 68975 (deposited May 6, 1992)) and has a specific location for the restriction enzyme Pvul. Then PCR was performed using the aforementioned primary chains and protoplasmic ptrphs BHP 1-13 as the basis for amplification of the modified BHP gene. The enlarged BHP gene was dissolved with the restriction enzymes Pvul and Saci and ligated with a DNA fragment obtained by dissolving the

protoplasmic ptrphs BHP 1-13 with the same enzymes to obtain the receiving vector of BHP. The receiving vector was used to transform E. coli. and the protoplasm containing the modified BHP gene is separated and is called ptrphs BGHRAN.

In order to introduce a trpA copy end gene into the BHR receiving vector, primary PCR strands were prepared including a trpA copy end gene with established localization for the restriction enzyme Sall and another for the restriction enzyme Pvul. PCR is then obtained by using the aforementioned primary chains and protoplasmic ptrphs BXR 1-13 as the basis for amplifying the DNA fragment comprising the trpA copy-finishing gene. The amplified DNA fragment was digested with the restriction enzymes Pvul and Sall and ligated with the DNA fragment, which was obtained by dissolving the protoplasmic ptrphs BGHRAN with the same enzymes to obtain the receiving vector of BHP. The receiving vector is used to transform E. coli as the protoplasm, including the modified BHP gene and the trpA copying finishing gene, is separated and called ptrp3H BGHRAN.

The receiving vectors of the present invention are used to transform an E. coli cell, for example, an E. coli W3110 (ATCC 37339), which is suitable for producing the BHP gene into the receiving vector. The transformed E. coli cell is cultured under a condition that allows the production of BHR, and the resulting BHR can be separated and purified from the culture according to any conventional method.

The present invention also provides a process for mass production of VHP, comprising culturing E. coli cells transformed with a receiving vector according to the invention, and separating VHP from the culture. Depending on the inventive process, it is possible to produce the HPP of E. coli transformant in an amount greater than 50% of the total amount of protein obtained.

The following examples are intended to illustrate the present invention without limitation in scope.

The percentages quoted below for solid in solid, liquid in liquid and solid in liquid are based on weight / weight, volume / volume and weight / volume respectively, unless otherwise indicated.

Example 1: Increasing a Modified BHP Gene and Building a Receiving Vector Incorporating the Same Gene (Step 1)

Random primary PCR chains corresponding to 5'-kr. VHR field and have the following nucleotide sequence are

-6 synthesized based on information from the nucleotide sequence of the BHP gene (8g. Korean patent N 92-3665):

PBGHRAN Primary Circuit:

5'-TGCTGAGCTCGNAGNACNGCRTTNGCRAANAGNCCNGANAGNGACATNGC NGGRAANGCCATTTATAATTCCTCCA-3 ', where N is A, T, G or C; and R is A or G.

The PBGHRAN primary strand includes a SacI established localization and a modified nucleotide sequence encoding 16 amino acids from the N-terminus of BHP.

A primary strand of PPBR3720 comprising a portion of the Atr gene in protoplasmic ptrphs BHP 1-13 (see Korean Patent No. 92-3665; ATCC 68975) and a Pvul specific location is also synthesized, their nucleotide sequence being as follows:

5 '- TCCTTCGGTCCTCCGATCGTTGTCA - 3' (Step 2)

Place 1 ng protoplasmic ptrphs BMP 1-13 in a test tube as a base, 2 g each of PBGHRAN and PPBR3720 primary chains prepared in step 1.10 1 of 10x polymerase reactive buffer (10 mM Tris-HCl, pH 8.3, 500). mM KCI, 15 mM MgCl ₂ , 0.1% (weight / volume) gelatin), 10 1 mixture of dNTR's (2 mM each of dGTP, dATP, dCTP and dTTP), and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, U .

SA) and distilled water to give a final volume of 100 l.

PCR was performed by repeating 25 times in the following cycle: 95 C for 1 minute (denaturation), 55 C for 40 seconds (heating), and 72 C for 2 minutes (polymerization).

The PCR product obtained in the above manner was subjected to 5% polyacrylamide gel electrophoresis and as a result confirmed that about 990 bp of DNA was increased. DNA is purified by the same polyacrylamide gel electrophoresis and is called BGHRAN.

(Step 3) g of protoplasmic ptrphs BMP 1-13 were completely dissolved with SacI in NEB (New England Biolabs Inc.) buffer 1 (10 mM Bis Tris propaneN1, 10 mM MgCla, 1 mM DTT, pH 7.0) at 37 C for 1 - 2 hours, then completely dissolved with Pvul in NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgC12, 1 mm DTT, pH 7.9) under the same conditions. The resulting mixture was

subjected to 0.7% agar gel electrophoresis to isolate about a 2 kb fragment, called the PBGH-T / P fragment.

g of the BGHRAN fragment obtained in step 2 was completely dissolved with SacI in NEB buffer 1, then completely dissolved with Pvul in NEB buffer 3. The resulting DNA fragment was extracted with phenol / chloroform and then dissolved in 20 l of TE buffer. The DNA fragment is called the BGHRAN-T / P fragment.

The following ligation reaction was performed using the DNA fragments already obtained: 100 ng of each of the BGHRANT / P and PBGH-T / P fragments, 2 1 of 10x ligation buffer and 10 units of T4 DNA were placed in the reaction vessel. ligase; distilled water was added to give a final volume of 20 1. The ligation was carried out at 16 C for 12 hours.

E. coli W3110 (ATCC 37339) was transformed with the ligation mixture to produce a recombined E. coli transformant containing the protoplasmic ptrphs BGHRAN including the BGHRAN fragment (see Figure 2).

Example 2: Preparation of a modified BHP gene and gene sequence (Step 1)

About 100 columns of E. coli transformant obtained in Example 1 were cultured in liquid Luria medium (6% Bactotrypton, 0.5% leaven extract, 1% NaCl) containing 50 g / ml ampicillin at 37 C for 12 hours. 3 ml of culture was transferred to 300 ml of M9 medium (40 mM K2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 18.7 mM NH4Cl, 1% glucose, 0.1 mM MgSO4, 0.1 mM CaCl2, 0.4% casino acid, 10 g / ml. vitamin Βμ 40 g / ml ampicillin); cultured by shaking at 37 C for 4 hours.

When the OD value of the culture at 650 nm reaches 0.3, indole acrylic acid (IAA) is added to the culture to reach a final concentration of 50 g / ml. After 4 hours, the OD of the resulting crop was determined and centrifuged at 11,000 rpm. per minute for 25 minutes to collect E. coli cell pellets. Cell precipitate is subjected to 15%

SDS-PAGE by application of the Aayemli method (Nature, 227, 680 (1970)) to confirm the production of BHR. Clones showing a 40% greater amount of BHR obtained compared to the E. coli transformant including protoplasmic ptrphs BHR 1-13 (control group) were harvested and the protoplasm was called ptrphs BGHRAN # 5 (see Fig. 3).

(Step 2)

The sequence of the BHP gene in the protoplasmic ptrphs BGHRAN # 5 obtained in step 2 is as follows: the protoplasmic ptrphs BGHRAN # 5 is completely dissolved with SacI in the NEB

buffer 1, then completely dissolved with EcoRI in NEB buffer 3. The reaction mixture was subjected to 7% polyacrylamide gel electrophoresis to separate the DNA fragment having about 187 bp, the latter being called the BGHRAN-T / R fragment.

On the other hand, 2 g of protoplasmic M13tr18 were completely dissolved with SacI in NEB buffer 1, then completely dissolved with EcoRI in NEB buffer 3. The resulting mixture was subjected to 0.7% agar gel electrophoresis to separate the DNA fragment, which is about 7 kb and is called the M13-T / R fragment. 100 ng of each of the BGHRAN-T / R and M13-T / R fragments, 2 l of 10x ligation buffer and 10 units of T4 DNA ligase were placed in a ligation reaction vessel; distilled water was added to a final volume of 20 1. The ligation was carried out at 16 C for 12 hours.

1 of the resulting ligation mixture was mixed with 200 l of E. coll JM105 (ATCC 47016) complete cells and 8 l of 0.2M IPTG solution, 100 l of pre-cultured auxiliary cells (E. coli JM105) were added to the mixture. , 3 ml of 2XYT agar (soft agar; 16 g Bactotrypton, 10 g yeast extract, 5 g NaCl, 5 g Bactoagar / l), and 25 1 of 4% X-gal. The mixture was applied to a Min A plate (10.5 g KH2PO4.1 g (N114) 2804, 0.5 g NaCl, 12 g Bactoagar, 1 ml 20% MgSO4, 0.5 ml 1% Vitamin B, 10 ml 50% glucose / l), and cultivated npu 37 C for 12 hours.

The clear plates formed in the medium were collected, transferred to 2 ml of 2XYT liquid medium, and then cultured at 37 C for 5 hours. The culture was centrifuged to give a precipitate and 1/4 volume of PEG / NaCl (20% polyethylene glycol, 2.5 M NaCl) was added thereto. M13 bacteriophage was separated from the supernatant; it produces one strand of DNA; the nucleotide sequence of the 5 'terminal field of the BHP gene was confirmed (see Fig. 1).

Example 3: Construction of a Receiving Vector Incorporating a TrpA Copy End Gene (Step 1)

The following initial strands were synthesized to amplify the trpA copy end gene and to incorporate it into the BMP receiving vectors prepared in Example 2:

PTRPATER Chain: 5'-GCTTTGTCGACTAATTAAAGCCCGCCTAATGAGCGGGCTTTTTTTTGCCT

CGCGCGTTTCGGT-3 '.

The PTRTATER chain includes an established localization for Sall and the 23rd-50th and 51st-67nd nucleotides correspond to the trpA copy end gene and to the nucleotide sequence after the 3'-end of the BHR gene in the vector, respectively.

Kakm

Protoplasmic PPBR3740: 5'-TGACAACGATCGGAGGACCGAAGGA-3 '(Step 2)

Place 1 ng of protoplasm ptrphs BMP 113 as a base in a test tube, 2 g each of PTRPATER and PPBR3740 primary chains prepared in step 1.10 1 of 10x polymerase reaction buffer (10 mM Tris-HCl, pH 8.3, 500 mM) KC1.15 mM MgCl ₂ , 0.1% (w / v) gelatin), 10 l of 2 mM dNTP (each of dGTP, dATP, dCTP and dTTP is 2 mM), and 2.5 units of Taq DNA polymerase; distilled water was added to give a final volume of 100 1. PCR was performed by repeating 25 times in the following cycle: 95 C for 1 minute, 55 C for 40 seconds, and 72 C for 2 minutes.

The resulting PCR product was subjected to 1% agar gel electrophoresis to confirm that about 1.5 kb of DNA was amplified. The DNA is purified by the same agar gel electrophoresis and is called a PBR fragment.

(Step 3) g of protoplasmic ptrphs BHP 1-13 was completely dissolved with Sall and Pvul in NEB buffer 3 and the resulting mixture was subjected to 0.7% agar gel electrophoresis to isolate a fragment of about 1.5 kb, called the PBGH-P fragment / L.

g of the protoplasmic ptrphs BGHRAN # 5 obtained in step 1 of Example 2 was completely dissolved with Sall and Pvul in NEB buffer 3 and the resulting mixture was subjected to 0.7% agar gel electrophoresis to isolate about a 1.5 kb fragment, called the PBGHRAN-P fragment / L. Subsequently, 2 g of the PBR fragment obtained in step 2 were completely dissolved with Sall and Pvul in NEB buffer 3. The resulting DNA fragment was extracted with phenol / chloroform and then dissolved in 20 l of TE buffer. The DNA fragment is called PBR-P / L.

Using the above DNA fragments, a ligation reaction was carried out as follows: 100 ng of each of PBGH-P / L and PBGHRAN-P / L fragments were placed in two tubes, respectively. To each tube were added 100 ng of PBR-P / L fragment, 2 l of 10x ligation buffer and 10 units of T4 DNA ligase; distilled water was added to give a total volume of 20 1. The ligation reaction was carried out at 16 C for 12 hours.

E. coli W3110 (ATCC 37339) cells were transformed with the ligation mixture in accordance with SaS1 ₂ method (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982) to obtain recombinant E. coli transformants containing protoplasmic ptrp H

- 10BXR 1-13 (tube A) or protoplasmic ptrp3H BGHRAN (tube B) (see Fig. 2).

Example 4: Preparation of BHR by Using Receiving Vectors Including a Modified BHR Gene and a TrpA Finishing Gene

The E. coli transformants obtained in Example 3 were cultured by shaking in liquid Luria medium (6% Bactotrypton, 0.5% leaven extract, 1% NaCl) containing 50 g / ml ampicillin at 37 C for 12 hours. 3 ml of culture was transferred to 300 ml of M9 medium (40 mM K2HPO4, 22 mM KH2PO4, 8.3 mM NaCl, 18.7 mM NH4CI, 1% glucose, 0.1 mM MgSC> 4, 0.1 mM CaCl2, 0.4% casino acid, 10 g (ml vitamin B ^, 40 g / ml ampicillin); cultured by shaking at 37 C for 4 hours.

When the OD value of the culture at 650 nm reaches about 0.3, indole acrylic acid is added to reach a final concentration of 50 g / ml. After 4 hours, the O. 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 minutes to collect E. coli cell swell. The cell swell was subjected to 15% SDS-PAGE according to the Laemli method (Nature, 227,680 (1970)) to confirm the obtained BMP. The result is shown in FIG. 3.

As shown in FIG. 3, the amounts of BHR produced by E. coli cells modified with protoplasmic ptrp3H BHR 1-13 or ptrp3H BGHRAN are 39.9% and 57.3%, respectively, based on the total amount of protein produced in the cell. These amounts are 40-50% higher than those produced by E, coli. transformed with protoplasmic ptrphs BHP 1-13 or ptrphs BGHRAN # 5, e.g. 26.3% and 39.9% respectively (see Figure 4).

E. coli W3110 Transformed with Plasma Protective Ptrp3H BGHRAN was deposited on 26 December 1994 at the Korean Collection for Taipei Kalchuz (CCTS) with an incoming number under the terms of the Budapest Agreement on the International Recognition of Microorganisms Deposits for Patent Procedures.

While describing the invention in terms of the above specific characteristics, it should be noted that various modifications and changes can be made to it by persons skilled in the invention, which changes also fall within its scope as defined by the claims.

Claims (7)

A receiving vector for bovine growth hormone comprising a modified bovine growth hormone gene, characterized in that the nucleotide sequence in its 5'-terminal field is modified without altering the amino acid sequence encoded therein and a trpA copy terminator gene inserted after the 3'-end of the gene. The receiving vector of claim 1, wherein the 5'-terminal field of the modified bovine growth hormone comprises the nucleotide sequence, as should: 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-', where ATG is the translated start codon of the BHP gene. The receiving vector according to claim 1, characterized in that the trpA copy finishing gene comprises the following nucleotide sequence: 5'-AGCCCGCCTA ATGAGCGGGC TTTTTTT-3 '. The receiving vector according to claim 3, characterized in that it is the protoplasmic ptrp3H BGHRAN (KTCC 0143BP). 5. E. coli cell transformed with the receiving vector according to any one of claims 1 to 4. An E. coli cell according to claim 5, characterized in that E. coli W3110 is transformed with the protoplasmic ptrp3H BGHRAN (KTCC 0143BP). A process for the mass production of bovine growth hormone, which comprises culturing an E. coli transformant according to claim 5, and separating the bovine growth hormone from the culture.