KR900002841B1 - Method for producing of humman insulin by genetic engineering - Google Patents

Abstract

In the method, using the synthetic method of solid phosphite the oligonucleotide coding for human insulin A and B was synthesized. From the synthesized oligonucleotides, the genes for A and B chain of insulin was constructed by the shot-gun method. After the E. coli plasmid, pUC19 was opened by the restriction enzyme Sph I and BamH I, and the above constructed gene was inserted by the reaction of DNA ligase. The inserted plasmid was called pSY A and pSY B. The base sequence of pSY A and pSY B was identified by the Sanger's method.

Description

New Gene Design of Human Insulin Using Genetic Engineering Technique and Preparation of Human Insulin Using It

1 is a schematic diagram of a gene designed to command a peptide of the insulin A chain.

Figure 2 is a schematic diagram of the gene designed to command the peptide of the insulin B chain.

FIG. 3 shows the results obtained by assembling chemically synthesized oligonucleotides into genes for insulin A chain or B chain, followed by polyacrylamide gel electrophoresis for denatured gene separation.

4 is an explanatory diagram illustrating a process of constructing plasmid pSYA or pSYB by inserting the assembled insulin A chain or B chain gene into E. coli plasmid pUC 19.

FIG. 5 is a part of the results of confirming the insulin A chain or B chain gene present in the plasmid pSYA or pSYB according to the base sequence determination method using dideoxynucleosides.

FIG. 6 is an explanatory diagram of a process for preparing pSYAP or pSYBP, which are plasmids constructed to be expressed by directly connecting an insulin A chain or B chain gene to a beta galactosidase promoter.

FIG. 7 is an explanatory diagram of a process for preparing pAA, pTA, pAB, and pTB, which are plasmids constructed by connecting the insulin A chain or B chain gene to the first half of the beta galactosidase gene and expressed as a fusion protein.

FIG. 8 is a photograph of microscopic observation of inclusion bodies accumulated in Escherichia coli by mass production of insulin A chain or B chain peptide (A: before expression, B: after expression).

The present invention provides a novel gene specifically designed and synthesized genes corresponding to A and B chains in human insulin structure to produce human insulin using genetic engineering techniques, and the synthetic genes are microorganisms and plants and animals. The present invention relates to a process for producing insulin by transforming and then expressing a cell.

Insulin is a hormone secreted by beta cells on the pancreatic islet of Langelhans, and its deficiency causes diabetes and hyperglycemia. When produced from a gene, it is produced in the form of pre-proinsulin, but 24 amino acids (pre-sequence) attached to the amino terminus of the B chain are removed by intracellular network tissue, which is a precursor of insulin. It is made of (pro-insulin). Proinsulin is composed of the A chain with 21 amino acids, the B chain with 30 amino acids, and the C chain with 35 amino acids connecting the two chains, which are separated by trypsin-like degrading enzymes. Only after quenching and releasing the C chain, it becomes a physiologically active insulin with only A and B chains.

Existing insulin production processes include separating and purifying animal insulin derived from animal organs. Insulin, present in all animals, is one of the most conserved proteins in the amino acid sequence, making this ideal application of animal insulin, but this is not only a complex process that requires many organs of the animal, There is a possibility that other types of hormones may be mixed in a very small amount and may also cause chronic illness in patients with immune responses sensitive to human insulin and a slightly different amino acid sequence.

Therefore, in order to solve these problems, it has been developed in a method of modulating the amino acid at the carboxyl terminus of the B chain where the amino acid sequence of animal insulin and inteinsulin are different from each other, and it is known. 79,898, Japanese Patent Laid-Open No. 60-70,100). In particular, after alanine (alanine) present in the B chain carboxyl terminal of the isoline isolated from porcine pancreas using a specific enzyme, it is hydrolyzed by a specific enzyme, and then threonine (threonine) at the B chain carboxyl terminal of human insulin Is replaced by However, the conversion rate of the hydrolysis and binding reaction by the enzyme is not only low, but the production process is much more complicated than the production process of animal insulin.

It is already known that insulin can be mass-produced by E. coli by recombining its genes, as the introduction of gene synthesis technology is inevitable to mass-produce human proteins or hormones (US Patent 4,366,246). , Japanese Patent Laid-Open No. 57-163,352). Genes used for such gene recombination are genes that command human proinsulin or genes that direct human insulin A and B chains, and these genes are inserted into a plasmid, which is a carrier, to transform E. coli. Later, it became known that a manufacturing process for expressing it and mass-producing the corresponding protein and converting it into insulin was developed. Among these, when the gene of human proinsulin is used, the process of converting the proinsulin produced by Escherichia coli into human insulin is in a situation which is not well established yet. Therefore, the process of mass-producing chain and B-chain peptides in Escherichia coli as a corresponding protein using human insulin A and B chain genes, and then combining the two chains by chemical methods has been most widely known. .

In the present invention, the human insulin A chain and B chain peptides are designed and synthesized so as to have a unique DNA sequence unlike those known in the art for mass production of microorganisms and flora and fauna cells. . That is, in the present invention, considering several specific matters in designing and designing genes of human insulin A and B chains, new genes having a corresponding DNA base sequence were synthesized and assembled.

In order to facilitate the introduction of the synthesized genes into microorganisms and flora and fauna cells, these genes were cassetteized by selecting and designing junctions of restriction sites or restriction sites of restriction enzymes specific to the sock ends of these genes. That is, by designing a junction point of the cleavage site of restriction enzyme Sph I at the gene site that commands amino acid methionine, which is at the amino terminus of the two-chain peptides and at the start of peptide biosynthesis, among the sites that command methionine in microorganisms and plant genes It is possible to replace these synthesized genes at sites that can be cleaved with restriction enzymes Sph I or its cleavage site with complementary restriction enzyme Nla III, so that it can be easily matched to the reading frame of the base code in the gene. In addition, the genes of the human insulin chains thus joined and replaced were easily expressed as a single peptide or a peptide in fused form. In addition, by designing recognition sites such as restriction enzymes Dde I, Sac I, Xho I and BamH I around the stop codon, the end of biosynthesis of the two chains, one of several restriction enzyme recognition sites in the gene to be replaced Restriction enzymes or restriction enzymes with junctions complementary to the cleavage sites of these restriction enzymes Cvn I (Dde I), Sal I, Ava I (Xho I), Mbo I, Bal II, Bcl I, Xho II (BamH I Easily combined with cutting parts such as In addition, in order to efficiently carry out this purpose, it is necessary to break away from the existing publicly known technology that has matched the codon usage of E. coli to the codon usage of E. coli for mass production of human insulin by genetic recombination technology. By maintaining the base code sequence of the insulin gene present in the human body, it was possible to express in any cell. It only affects the sequence of amino acids that can be dictated by the sequence of guanosine, which can be a problem in the synthesis of oligonucleotides, which are single-stranded fragments of the human insulin gene. The base sequence was adjusted within the range. The genes of human insulin A and B chains designed and designed in consideration of these points are the same as those of Fig. 1 and Fig. 2.

In the present invention, in order to synthesize and assemble the gene of human insulin designed as described above, as shown in FIGS. 1 and 2, the genes of the A chain and the B chain are chemically synthesized after dividing into six oligonucleotides each of 6 fragments. These fragments were assembled by biochemical methods. In dividing genes designed to command the human chain A and B chains, the cleavage positions were selected to minimize complementarity between the oligonucleotide itself and each other. In order to avoid complementary duplication, the base sequence number of the junction was 6. The oligonucleotides of the human insulin A chain and B chain genes thus designed and synthesized were assembled by DNA binding enzymes (T4 DNA ligase) to assemble into insulin chains and chain genes.

In the present invention, the assembled gene may bind to DNA carriers, such as plasmids, bacteriophages, or flora and fauna viruses to transform microorganisms and flora and fauna cells. Here, the plasmid of E. coli was inserted into pABG (Korean Patent Application No. 14,289,1987) or pTBG (Korean Patent Application No. 14,287,1987) having a PL promoter or tac promoter. In the present invention, in order to express the genes of insulin A and B chains by themselves, an attempt was made to directly link them with a promoter, and also beta galactosidase (linked to these promoters to produce a fused protein) Connection with the first half of the beat glactosidase gene (lacZ) was also performed. The plasmids thus constructed were able to confirm the biosynthesis of peptides in each chain by attempting transformation in E. coli.

Example 1

Chemical Synthesis of Oligonucleotides Comprising Human Insulin A and B Chain Genes The automated human phosphite synthesis is adopted in the synthetic method of DNA synthesis. And oligonucleotides, which are DNA fragment manipulations in which the genes instructing the B chain are divided, are synthesized. Accordingly, the synthesis direction progressed from the 3 'end to the 5' end. At this time, the first base used was attached to the silica solid phase, the reaction size of the attached base was 1 micromolar. Four consecutive chemical reactions were carried out to attach the following bases. The reactions consisted of detritylation, condensation, oxidation and capping. In the Tam protection reaction, 3% was treated with acetic acid chloride solution to expose the hydroxyl groups at the 5 'end, and the orange color of the trityl group produced as a by-product of the reaction indicated the amount of base attached to the silica solid. The hydroxyl group of the deprotected base and the phosphosamidite form of the next base were reacted in the presence of tetrazole, and one base was used to attach the next one base. Since the base bond formed in the condensation reaction is a phosphite bond, it must be oxidized and transformed into a phosphate bond, which is a natural base bond. For this purpose, a 1% iodine solution dissolved in a mixed solvent of pyridine and acetic acid is treated. Oxidized. In addition, in order to prevent the reaction from proceeding in the non-condensed base in the condensation reaction, the hydroxyl group of the base exposed in the previous deprotection reaction was capped by acetic acid with acetic anhydride in the presence of dimethylaminopyridine. Through this process, the solid silica to which one base was attached was subjected to deprotection reaction, condensation reaction oxidation and capping reaction, and the following bases were sequentially attached. Oligo synthesized by attaching to the last base of each oligonucleotide constituting the genes of the insulin A chain and chain B according to FIGS. 1 and 2, recovering the solid silica and treating it at 50 ° C. using concentrated ammonia water. Benzoyl and isobutyl groups that protected the nucleotides from the silica support and protected the amino groups of adenosine guanosine and cytosine that protected the phosphate bonds between bases ) Was decomposed. The oligonucleotide finally synthesized was subjected to electrophoresis on 6-15% denatured polyacrylamide gel to separate the unreacted materials, and the Sep-Pak column was again separated. Desalination purification.

Example 2

Gene Assembly of Insulin A and B Chains from Synthesized Oligonucleotides

Oligonucleotides synthesized by dividing the genes that command the insulin A and B chains into six groups as shown in FIGS. 1 and 2 are assembled into genes of the chains by a biochemical method using a DNA binding enzyme (T4 DNA ligase). It became. First, for easy tracking and quantification of each oligonucleotide synthesized in the examples, oligonucleotides (i-9 and i-13 in FIG. 1 and I-3 in FIG. 2) to be located at the 5'-end of the entire designed gene The 5'-terminal hydroxyl groups of all oligonucleotides except for and I-8) were labeled with radioisotopes. The radioisotope was labeled with a phosphorus (32p) with a mass of 32 at the gamma position of adenosine triphosphate (ATP), using a DNA kinase (T4 DNA kinase) for 1-2 hours at 30-40 ° C. In response, a pregnant group with radiation of adenosine triphosphate was transferred to each oligonucleotide. Thus, each radioactive oligonucleotide was passed through a Seppect column to remove unreacted adenosine triphosphate, and then adjusted to the same amount by measuring radioactivity. As a method of assembling oligonucleotides in gene units, there are block recipes, shot-gun assembly methods, and continuous assembly methods. Since the lengths of the A and B chain genes of insulin are relatively short, In the present invention, a shotgun assembly method was used.

Each of the oligonucleotides labeled above with radioactivity was mixed to the same amount, and oligonucleotides located at the 5'-end of each gene left unradioactively were added and heated at 100 ° C. for at least 2 minutes, and left at room temperature. . The mixed solution of oligonucleotides was assembled into genes of the insulin A and B chains by reaction at 4-40 ° C. for 1-50 hours by a biochemical method using a DNA binding enzyme. Finally, the assembled genes were subjected to electrophoresis on 5-10% polyacrylamide gel for denaturation of the degenerate gene, and then exposed and photosensitive to the X-ray film to confirm the gene position of each chain (FIG. 3). Only the regions corresponding to these genes were cut out, eluted in buffer, and precipitated in ethanol to recover only these genes.

Example 3

Insertion of the assembled insulin A and B chain genes into plasmids and transformation of E. coli using the inserted plasmids

PUC 19, a plasmid of Escherichia coli, was cleaved with restriction enzymes SphI and BamH I and inserted into the A- or B-chain gene. Restriction enzymes Sph I and BamHI were added at the same time and reacted for 30 minutes to 2 hours at 30-40 ° C. to decompose and cleave plasmid pUC 19, extract impurities with phenol and chloroform, and precipitate and purify ethanol. To the recovered plasmid fragment, 2-10-fold genes of the insulin A chain and the B chain assembled and purified in Example 2 were added, and then reacted and inserted in the presence of a DNA binding enzyme at 4-40 ° C. for 2-40 hours. As shown in Figure 4, the plasmid in which the gene of insulin A chain was inserted and replaced in plasmid pUC 19 of the plasmid thus prepared was named pSYA, and the plasmid in which the gene of insulin B chain was inserted and replaced was named pSYB, respectively.

Plasmids pSYA or pSYB replaced by inserting the genes of the insulin A chain or B chain were transformed into E. coli strain JM103 (Esch-erichiacoli JM103), and then, E. coli carrying these plasmids were selected and separated from the selection medium. For transformation, a method known by D. Hanahan (DNA Cloning, Vol. 1, pp. 109-135, 1985 edition) was used. Based on the fact that the gene of the insulin A chain or B chain is inserted into the E. coli plasmid pUC 19, the beta galactosidase gene is inactivated, and thus 5-100 micrograms of ampicillin per milliliter of the medium ( ampici-llin), 0.01-1.0 mmol of isopropyl-thiogalactopyrano-side (IPTG) and 0.001-0.01% 5-bromo-4-chloro-3-inmolyl-gal Escherichia coli forming white colonies was isolated by using LB medium containing lactopyranoside (5-bromo-4-chloro-3-indolyl-galactopyranoside; X-gal) as a selection medium. Thus, the plasmid is isolated and purified from the E. coli strains selected according to a known method, and then digested with restriction enzymes EcoR I, BamH I, Hind III, Pvu II, etc., to the desired direction at the desired position of the insulin chain A or B chain. It was confirmed whether the plasmid pSYA or pSYB inserted into.

Example 4

Identification of the base sequence of the insulin A chain or B chain gene inserted into the plasmid pSYA or pSYB

Oligonucleotides (5'-ACAACGTCGT GACTGGGAAAACCC-3 '5'-GCTATGAC CATGATTACGCCA-3') having a nucleotide sequence around the position where the insulin A chain and B chain genes are inserted in the plasmid pSYA or pSYB are used as primers. Inserted insulin according to the method of determination of nucleotide sequence using a dideoxyuncleoside known by F. Sanger et al. (Proceedings of National Academic Science, USA, Vol. 74, pp. 5,463, 1977). The nucleotide sequence in the A-chain or B-chain gene of was confirmed. After denatured plasmid pSYA or pSYB purified in Example 3, and denatured in the presence of DNA polymerase (DNA poymerase I, Klenow frgment) DNA elongation was extended by adding a mixed solution of deoxynucleoside triphosphate (ddNTP) and dideoxynucleoside triphosphate (dNTP). In other words, a label labeled with phosphorus (32p) with a mass of 32 at the alpha position of deoxyunclesoide triphosphate (dATP) was used to label the reaction with radioactive elements. The reactants reacted by the polymerization reaction were electrophoresed on polyacrylamide gel for 5-10% separation of the modified gene, and the gel was exposed to and exposed to an X-ray film to determine the base sequence according to the position of each band. 5 is a part of determining the nucleotide sequence of the insulin A chain or B chain gene inserted into the plasmid pSYA or pSYB, it can be seen that the same as designed and designed in Example 1

Example 5

Plasmid Construction Engineered for Easy Gene Expression and Production in Escherichia Coli for Production of Insulin A and B Chain Peptides

To express the genes of insulin A chain or B chain in Escherichia coli, these genes must be linked to strong promoters, and furthermore, when the fusion protein is to be produced, the unit of interpretation of the genes must be identical, and the gene product In order to maximize production, gene expression must be able to be induced.

First, in order to express the genes of the insulin A and B chains, the plasmid pSYA or pSYB constructed in Example 3, which was already linked to the promoter of beta galactosidase, was manipulated to match the interpretation units. That is, these plasmids were hydrolyzed with restriction enzyme HindIII, and then partially hydrolyzed with another restriction enzyme NlaIII and eluted plasmid at the same site as the original plasmid on agarose gel. The plasmid fragment thus obtained was bound again in the presence of DNA binding enzyme, introduced into E. coli strain JM 109, and the plasmids were separated to select that the recognition site of restriction enzyme Hind III was lost. This resulted in the loss of 19 base pairs in the plasmid pSYA or pSYB and the rebound plasmids named pSYAP or pSYBP, respectively (Fig. 6).

In addition, the beta galactosidase gene (lacZ) obtained by cleaving the plasmid pORFl by the restriction enzymes Pst I and BamH I and the plasmid pORFl by cleavage by the two restriction enzymes Pst I and BamH I Plasmid pABG (Korean Patent Application No. 14,287,1987) prepared by linking a fragment containing a DNA binding enzyme and plasmid pT-6 with the restriction enzymes Pst I and Sal I and having a Tac promoter region. The plasmid pTBG prepared by linking the fragment with the beta galactosidase gene region obtained by cleaving the plasmid pABG by restriction enzymes Pst I and Sal I by separating the fragments in the presence of DNA binding enzyme (Korean Patent Application No. 14,289,1987) ) Was used as a carrier for gene expression in fused form.

In order to produce the insulin A chain or B chain peptide fused with beta galactosidase, the gene of each chain of insulin must be combined into the beta galactosidase gene, in which case the plasmid pABG or pTBG is restricted to the restriction enzyme BamH I. After hydrolysis with and Ava I, the first half of the beta galactosidase gene having 1,327 base pairs was cleaved and separated, and then partially hydrolyzed with restriction enzyme Nla III to separate and prepare a gene fragment having 1,076 base pairs. In addition, the insulin A chain or B chain gene present in the plasmid pSYA or pSYB prepared in Example 3 was hydrolyzed by restriction enzymes Hind III and Sst I and hydrolyzed by restriction enzyme Nla III to assemble in Example 2 Separated out. The fragments containing the first half of the beta galactosidase gene dominated by the Piel promoter or Tak promoter thus obtained are combined with the genes of the respective insulin chains separated above in the presence of DNA binding enzyme, and the bound gene fragments are separated. I did it. The fragments thus assembled and isolated were inserted into the original plasmid pABG or pTBG using DNA binding enzymes for replication and expression in Escherichia coli. In this case, the plasmid pABG obtained by hydrolysis with restriction enzymes BamH I and Sst I or pTBG was inserted into a large fragment, wherein the plasmids into which the insulin A chain gene was inserted were named pAA and pTA, respectively, and the plasmids into which the insulin B chain gene was inserted were named pAB and pTB, respectively (FIG. 7).

In the reaction of the restriction enzymes used in the above plasmid construction process, 0.1-50 units per microgram of DNA was carried out at 30-40 ° C. for 30 minutes to 2 hours. Electrophoresis was performed after electrophoresis on the gel (agarose gel), and for the ligation of these gene fragments, DNA binding enzymes were used at 1-40 units per microgram of DNA for 10-100 hours at 4-40 ° C. Reacted. The plasmids thus obtained were transformed into Escherichia coli strain JM 109 as in Example 3, followed by isolation of the proliferative strains forming a white colony, separation and purification of the plasmids introduced into these strains, and then the plasmids of several restriction enzymes. Hydrolysis was performed to confirm whether the desired gene was inserted at the desired position. In particular, in the case of pSYAP and pSYBP plasmids, the recognition sites of restriction enzyme Hind III were identified. In the case of plasmids pAA, pTA, pAB, and pTB, the recognition sites of restriction enzyme Ava I were confirmed. FIG. 8 shows that the peptides of the chains A and B of insulin were produced in Escherichia coli by the plasmids thus constructed to form an inclusion body.

Example 6

Isolation of Insulin A and B Chain Peptides from E. Coli Constructed by Genetic Engineering and Assembly into Human Insulin

Escherichia coli transformed with plasmids pYAP, pAA, pTA (plasmid having an insulin A chain gene), pSYBP, pAB, and pTB (plasmid having an insulin B chain gene) constructed in Example 5 were loaded with ampicillin. Inoculated in Elbi (LB) medium added 5-100 micrograms per liter, and incubated at 37 ° C., and then the insulin A chain or B chain was isolated from the inclusion body in Escherichia coli according to a known method (Japanese Patent Publication No. 57-163,352). After separation and purification, these two chains were formed to form disulfide bonds according to a known method (US Pat. No. 4,421,685) to finally prepare human insulin. The human insulin thus produced was analyzed by high performance liquid chromatography and showed the same chromatogram as the insulin standard. As the analysis conditions used at this time, 50-50 mol of sodium acetate (buffer (pH 7.0) 20-50% isopropanol (isopropanol) was injected at a concentration of 0.5% per minute while flowing out at a rate of 2 milliliters per minute.

Claims (19)

The gene is designed to command the human chain A of human insulin and has the same junction point as the cleavage site of restriction enzymes at both ends to facilitate the insertion and expression of the gene by cassetteization, and the restriction enzyme Sph I at the 5'-end. It has the same junction point as the cleavage site, has a recognition site of restriction enzymes Xho I and BaMH I at the 3'-end, and the base sequence of the gene which commands the insulin A chain is the same as the base sequence of the insulin gene in the human body. Gene characterized in that it has an array. 5'-GGC ATT GTG GAA CAA TGC TGT ACC AGC ATC TGC TCC-3 ', 5'-CTC TAC CAG CTG GAG AAC TAC TGC AAC TAG-3', 5 '-TGA GCA CGA G-3', 5'-GAT GCT GGT ACA GCA TTG TTC CAC AAT GCC CAT G-3 ', 5'-GCA GRA GTT CTC CAG CTG GTA GAG GGA GCA-3', 5'-GA TCC TCG AGC TCA CTA CTT-3 ' It is a gene designed to command human chain B chain of human insulin, and it has the same junction point as the cleavage site of restriction enzymes at both ends, so that it can be inserted and expressed by the cassette, and the restriction enzyme Sph I is cleaved at 5'-end. Insulin gene having the same junction point as the site, the recognition sites of restriction enzymes Dde I, SacI, Xho I, and BamH I at the 3'-end, and the nucleotide sequence of the gene that commands the insulin B chain Gene to have the same arrangement as. 5'-TTT GTG AAC CAA CAC CTG TGC GGC TCA CAC CTG GTG GAA GCT CTC-3 ', 5'-TTC CAC CAG TGA GCC GCA CAG GTG TTG GTT CAC AAA CAT G-3 ', 5'-TAC CTA GTG TGC GGG GAA CGA GGC TTC TTC TAC ACA CCC AAG ACC-3', 5'-GGG TGT GTA GAA GAA GCC TCG TTC CCC GCA CAC TAG ATG GAG AGC-3 ', 5'-TGA GCT CGA G-3 ', 5'-GA TCC TCG AGC TCA GGT CTT-3'. A plasmid engineered to directly connect the gene of claim 1 or the gene of claim 3 with an expression promoter in the plasmid to produce an insulin A chain or B chain peptide itself, and the expression promoter is a promoter of beta galactosidase. Plasmid. Plasmid PSYAP in which the gene of insulin A chain was inserted into plasmid PUC 19 in the large fragment of plasmid PABG or PTBG obtained by hydrolysis with restriction enzymes BamH I and Sst I. E. coli strain transformed to have the plasmid of claim 6. A method for producing human insulin by extracting and separating the insulin A chain peptide and the insulin B chain peptide from the culture medium of E. coli culture according to claim 7, and then connecting the two chain peptides by disulfide bonds. The plasmid engineered to produce peptides of the insulin A chain by combining the genes of the protein already expressed in accordance with the interpretation unit in order to express the new gene as a fusion protein. The plasmid of claim 9 constructed by linking with a part of the first half or a part of the second half of the beta galactosidase gene to be expressed. The plasmid of claim 16, wherein the entire gene is controlled by the PI promoter when linked as part of a gene. Plasmid PAA or PTA with the gene of insulin A chain inserted. E. coli strain transformed to have the plasmid of claim 11. A plasmid that has been engineered to produce the insulin B chain by combining a part of a gene that is already expressed and an analysis unit in order to express the novel gene of claim 3 as a fusion protein. The plasmid of claim 21 constructed by linking with a part of the gene of the first or second part of the gene for expression as part of beta galactosidase. The plasmid of claim 21, wherein when linked as part of a gene, the entire gene is governed by the PI promoter or Tak promoter. Plasmid PAB or pTB with the gene of the insulin B chain inserted. The plasmid transformed E. coli strain of claim 17. The protein obtained by culturing Escherichia coli of claim 13 is isolated from the culture medium in which the insulin A chain is fused, and the fusion protein in which the insulin B chain is fused is purified from the culture medium in which Escherichia coli is cultured according to claim 25, followed by fusion of these two fusion proteins. A method of producing human insulin by cleaving a protein with bromine cyanide to purely separate the insulin A chain or the B chain, and connecting the two chain peptides by disulfide bonds.

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