KR930001386B1 - Method for preparation of epidermal growth factor gene - Google Patents

Abstract

Prodn. of human epidermal growth factor (EGF) comprises (a) construction of a vector contg. a DNA sequence that encodes the formation of a fused polypeptide of EGF gene attached through CP protein gene which are specific to staphylococcus protein A; (b) transfecting a microbial host with this vector; (c) propagation of the transformed microorganism; and (d) isolation of EGF from the recombinant fusion protein by cleaving it with blood coagulation factor Xa. EGF is a therapeutic for promoting the healing of wounds, burns, abrasions, gastric ulcers, etc..

Description

Method of manufacturing human epidermal growth factor by genetic recombination technology

Figure 1 shows the human epidermal growth factor (EGF) and the sequence of the nucleotides encoding the linking sites and restriction enzyme sites (indicated under the nucleotides), respectively, where the cleavage site by blood coagulation factor Xa is indicated by an arrow.

2 is a schematic of the method for preparing plasmid pRHC-EF.

3 is a result of polyacrylamide gel electrophoresis after treatment of the isolated CP polypeptide and epidermal growth factor fusion protein with activated blood coagulation factor Xa, where arrows indicate epithelial growth factor and CP polypeptide, respectively. 2 represents molecular weight standard.

4 shows the results of quantitative analysis of amino acid composition of recombinant epidermal growth factor finally separated by Protein A affinity chromatography.

5 is a schematic of the separation and purification process of human epidermal growth factor.

6 shows the sequence of 12 oligonucleotides synthesized.

The present invention synthesizes a gene of human epidermal growth factor using recombinant DNA technology, and inserts it into an expression vector to effectively prepare and easily isolate and purify epidermal growth factor using pRHC-10 expression vector in Escherichia coli. It is about.

Epidermal Growth Factor (Epidermal Growth Factor) is a 53 amino acid polypeptide known as mitogen that promotes the division of a large number of meschymal cells. The exact role in vivo is not yet known, but it has been found that there are various activities as follows.

In other words, the effects of epidermal growth induction, gastric acid secretion, hair growth inhibition, differentiation promotion, skin promotion, etc. are shown by the administration of epidermal growth factor. In response to various studies on physiological activity and clinical trials, mass production of epidermal growth factors has been attempted in various ways.

Mass production by the conventional peptide synthesis method has proved to be ineffective due to low efficiency of binding three disulfide bonds in epidermal growth factor to have physiological activity.

In addition, when the gene of epidermal growth factor was expressed directly in E. coli by gene recombination, it was reported that this polypeptide was easily degraded by protease in cells (Taniyama et al., J. Takeda Res. Lab., 45, 136). , 1986).

Therefore, a number of production methods using expression vectors that secrete epidermal growth factors out of cells have been reported to protect against proteases.

The present invention is a method for producing human epidermal growth factor in Escherichia coli in the form of fusion protein by gene fusion known to have better expression effect than the method of secreting it out of the cell, the gene for the structural protein used for gene fusion Fusion of a specific region (eg, Fc region) of immunoglobulin G, which specifically binds to Staphylococcus protein A, using protein engineering and fusion expression using a gene encoding the modified polypeptide The vector pRHC-10 can be used to easily purify the purified protein from the expressed fusion protein.

In addition, the gene encoding the human epidermal growth factor is determined by the common use of Escherichia coli based on the amino acid sequence of human epidermal growth factor (Gregory H., Natrue, 257, 325, 1975). codon) was used, and the DNA sequence was effectively expressed in microorganisms and had no problem in synthesis.

The expression vector used in the present invention is a tac promoter and a fusion gene in which the human epidermal growth factor gene is fused in frame to the 3 'end of the gene encoding the structural protein or CP polypeptide. Engineered for efficient expression by a lac I repressor. This expression vector can induce the expression of a gene using IPTG, an expression inducer, using a host cell, which is Lock I _q . The expressed fusion protein was dissolved in a suitable buffer solution, and the activated human coagulation factor Xa was used to cut out exactly the mature human epidermal growth factor from the fusion protein. Way.

The present invention is explained in more detail by the following examples.

Example 1

Twelve oligonucleotides (Ef-1-Ef-12) (see FIG. 6) consisting of 20-45 DNA bases and five bases at both ends complement each other by an automated phosphoamidite method. After synthesis and purification thereof as described below, a double-stranded DNA encoding human epidermal growth factor was prepared by combining the method described in Example 2. The synthesized oligonucleotides were extracted and purified by precipitation with ethanol.

Purified oligomers were separately purified by the desired oligomer by 8-16% polyacrylamide gel electrophoresis.

Example 2

100 picomolar oligomers were dissolved in 20 μl of each kation buffer (70 mM Tris pH 7.6, 10 mM, MgCl ₂ , 1 mM ATP, 0.2 mM Spermidine, 0.5 mM DTT) and 2 μl of T _4. The reaction was stopped by adding polynucleotide kinase and reacting at 37 ° C. for 30 minutes and heating at 85 ° C. for 10 minutes.

Phosphorylated oligomers and non-phosphorylated oligomers were each mixed with 0.9 picomolar and heated to 90 ° C. and then cooled very slowly to anneal as shown in FIG. 2.

5 μl of 10-fold concentrated ligation buffer (50 mM Tris pH 7.6, 10 mM MgCl ₂ , 20 mM DTT, 1 mM ATP) was added and T ₄ DNA ligase was added and reacted at 15 ° C. for 4-10 hours. The reaction compound was electrophoresed on a 1.5-2% agarose gel, and the epithelial growth factor gene DNA band corresponding to 199 bp was cut out under UV light and separated and purified by gel elution.

Example 3

Plasmid pUC 19 (obtained from KCTC) was digested with Xbal restriction enzyme, mixed with the gene encoding the extracted purified epidermal growth factor, T ₄ DNA ligase was added and reacted at 15 ° C. for 8 hours under ligation buffer. The reaction was transformed into E. coli HB101 strain (Bayer et al., J. Mol. Biol., 41, 459, 1969) treated with CaCl ₂ and then ampicillin in LB medium containing ampicillin (59 mg / ml). Strains with resistance were selected and each plasmid DNA was isolated to identify the plasmid into which the human epidermal growth factor was inserted to prepare a pUC-EF plasmid (see FIG. 2).

5 μg of this pUC-EF plasmid was digested with Xbal, and agarose gel electrophoresis was used to isolate and purify the 0.2 kbp human epidermal growth factor gene fragment. The pRHC-10 expression vector was partially digested with Xbal, followed by 0.2 kbp DNA fragment and T ₄ Ligation with DNA ligase produced the plasmid pRHC-EF as in FIG. At this time, the plasmid pIN-III (FIG. 2) used for preparing plasmid pRHC-10 was easily obtained from KCTC.

The fusion of epidermal growth factor and gene in frame (CP gene), which is a structural gene, was confirmed by analyzing DNA sequencing by the diedeoxy method.

Example 4

E. coli JM109 strain was transformed with plasmid pRHC-EF and pRHC-EF / JM 109 strain having ampicillin resistance was selected to confirm plasmid DNA. The recombinant strain was incubated in a 10 L fermenter at 37 ° C. until A ₅₅₀ = 0.3, and then IPTG (2 mM) was added to induce the expression of the cloned gene. After 5-6 hours of incubation, cell pellets were obtained by centrifugation. The cell precipitate was mixed well with 40 ml of buffer solution (50 mM Tris, pH 8.0, 25% glycerol, 1 mM EDTA), and then 100 mg of lysozyme was added to destroy the cells, followed by centrifugation to separate the inclusion body. Separated.

This inclusion body was washed with 0.5-1% Triton X-100 containing buffer (10 mM Tris, pH 8.0, 1 mM EDTA) and then dissolved in 8M urea buffer completely and passed through a Sephacryl S-200 (Sephacyrl) column. Fractions containing the fusion protein of epidermal growth factor were identified by polyacrylamide gel electrophoresis and collected and dialyzed at 4 ° C. for 24 hours in a buffer solution (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA). 2 ml of active coagulant factor Xa immobilized on sepharose-4B was added to the dialyzed solution to cut the junction between epidermal growth factor and CP protein, and the reaction was centrifuged to remove immobilized active coagulation factor Xa. After dialysis in 20mM ammonium acetate solution and confirmed the CP polypeptide and human epidermal growth factor cut by 18% SDS polyacrylamide gel electrophoresis method (Figure 3). The reaction was added to a Protein A affinity chromatography column and the column was washed continuously with 20 mM ammonium acetate buffer until the absorbance (A ₂₈₀ ) of the fractions passed through the column was near zero, followed by 0.2 M ammonium acetate buffer. Epithelial growth factor bound to protein A was eluted and the separated fractions were collected, dialyzed in 0.1 M acetic acid and lyophilized. After the final separated sample was hydrolyzed at 110 ° C. for 15 hours in 6N hydrochloric acid, the amino acid composition was confirmed by the CKB4150 amino acid quantitative analyzer. As a result, it was confirmed that the epidermal growth factor was correctly cut (see FIG. 4). It was confirmed by the Lowry protein quantitative method that the epidermal growth factor can be obtained in a high yield of about 15.3 mg from 11 cell embryos according to the process illustrated in FIG.

As can be seen from the above results, the epidermal growth factor sensitive to protease attack was protected from E. coli by proteolytic protease, so that E. coli cells could be stabilized. It has an affinity so that human epidermal growth factor can be easily separated from CP protein.

This principle of the present invention is designed to be applicable to any protein other than epidermal growth factor.

Claims (10)

Fusion of DNA sequences comprising genes encoding human epidermal growth factor (EGF) linked to genes encoding CP proteins capable of specifically binding to Staphylococcus protein A under transcriptional control in vectors suitable for host cells A method for producing a human epidermal growth factor characterized by efficiently producing a fusion protein in a host cell using an expression vector, and separating and purifying the human epidermal growth factor from the fusion protein. The human epidermal growth factor of claim 1, wherein the linking polypeptide of the CP protein and the human epidermal growth factor is a vector comprising a nucleotide sequence encoding an amino acid sequence that can be cleaved specifically by blood coagulation factor Xa. Preparation method (Fig. 1b). The method of claim 1, wherein the nucleotide sequence encoding the amino acid terminus is a nucleotide sequence encoding 120 amino acids in a proteolytically modified immunoglobulin Fc region. The method of claim 1, wherein the vector comprises a nucleotide sequence linked to a translation stop signal immediately after the nucleotide sequence encoding the expressed EGF. The method for producing human epidermal growth factor according to claim 1 or 4, wherein the synthetic nucleotide encoding the EGF is a vector having the following nucleotide sequence (FIG. 1a). AAA TCC GAC TCC GAA TGC CCG CTG TCC CAC GAC GG TGG TGC CTG CAC GAC GGT GTT TGC ATG TAC ATC GAA GCT TTG GAC AAA TAC GCA TGC AAC TGC GTT TGG GGT TAC ATC GGT GAA CGC TGC CAG TAC CGC AAC CTG AAA TGG TGG GAG CTC CGC The method of claim 1, wherein the transcriptional activation sequence is a vector which is a tag transcriptional activation sequence. The method of claim 1, wherein the recombinant DNA expression vector is a plasmid. The method of claim 1, wherein the host is Escherichia coli. The method for producing human epidermal growth factor according to claim 1 or 2, wherein the linkage region between the CP protein and the human epidermal growth factor is cut by blood coagulation factor Xa. The method for producing a human epidermal growth factor according to claim 1, wherein the immobilized activated coagulation factor Xa is used to facilitate the safety of the enzyme and to easily remove the enzyme.