CN1231259C - Generation of human insulin - Google Patents

Abstract

An improved and efficient method for producing recombinant human insulin by folding proinsulin hybrid peptides is provided.

Description

production of human insulin

This is a continuation of US serial application 08/175298, filed December 29,1993.

Background of the invention

In the description of the present invention, various documents are listed with Arabic numerals in parentheses. All citations of these references can be found after the specification and before the claims. All disclosures of these documents are incorporated by reference into this specification in order to more fully describe the state of the art to which this invention pertains.

Insulin, a polypeptide hormone necessary for the control of glucose metabolism, is administered daily to patients with diabetes, a metabolic disorder caused by insufficient supply of insulin.

In vivo, the hormone is first synthesized as a long precursor molecule and subsequently processed into its biologically active form consisting of A and B chains. In more detail, in the beta cells of the endocrine pancreas, the gene for preproinsulin is transcribed into pre-mRNA, which is then spliced to produce mature mRNA. The mRNA is translated into preproinsulin ( _NH2 -proregion-B-chain-C-peptide-A-chain-COOH), then reprocessed into proinsulin and finally into insulin. The first step in this process is the proteolytic removal of the prodomain, a hydrophobic signal sequence that transfers new strands across the microsomal membrane of the endoplasmic reticulum. In human preproinsulin, the length of the proregion is 24 amino acids.

In proinsulin, two regions of the polypeptide chain that will become mature insulin, the B- and A-chains are connected to each other by a C-peptide (or C-chain) that contains two pairs of basic amino acids at the N and C-terminal . In most C-peptides these pairings are Arg-Arg and Lys-Arg. Human C-peptide, including two side pairs of basic amino acids, contains 35 amino acids. The C-peptide is linked to both parts of the polypeptide to facilitate disulfide bond formation between the B and A fragments. Therefore, the action of C-peptide is largely independent of its structure. In fact, replacement with shorter synthetic bridges still folds the proinsulin molecule properly (1, 2).

Proinsulin folds with simultaneous oxidation of two interchain disulfide bonds and one A chain disulfide bond. During the final stage of maturation, proteolytic enzymes cleave at basic amino acids to release C-peptide and form mature insulin (3). In human insulin, the A chain has 21 amino acids and the B chain has 30 amino acids.

The annual demand for insulin in the world exceeds several tons, and the supply is seriously in short supply. Insulin has traditionally been produced from limited animal sources, primarily bovine and porcine pancreas products, which differ from human insulin and can therefore elicit harmful immune responses.

Research done in the sixties showed that insulin could be produced in vitro. Insulin is synthesized by combining the A and B chains in its S-sulfonated form (4) or by spontaneous reoxidation of reduced proinsulin (5). The latter method cannot be used for large-scale production of insulin due to the low protein concentration in the oxidation mixture. Insulin can be recovered after treatment with trypsin and carboxypeptidase B (6).

More recently semi-synthetic and biosynthetic (recombinant) human insulins are also available. Semisynthetic human insulin is produced from porcine insulin by trypsin-catalyzed exchange of Ala at position 30 of the B chain for Thr (the only difference between porcine and human insulin). Recombinant human insulin produced in E. coli or yeast will eventually replace all other production routes.

Semi-synthetic recombinant human insulin is now produced by two routes: the A and B chains are produced separately in E. coli and then joined together (7, 8), or enzymatically converted in E. coli (1, 8) or yeast ( 2, and polypeptides such as proinsulin expressed in 9).

In most cases, proinsulin is produced as a hybrid protein that accumulates as intracellular precipitated protein. The hybrid is purified normally and then cleaved with CNBr to release the proinsulin polypeptide. The latter is then modified by oxidative sulfitololysis to obtain proinsulin S-sulfonate. Proinsulin S-sulfonate is then purified and folded under reducing conditions to give proinsulin (8). Proinsulin is converted to insulin by the combined action of trypsin and carboxypeptidase B (6).

Patent publication EP195691 (Novo Nordisk A/B) describes proinsulin with the molecular formula B-Lys-Arg-A and its use for the preparation of insulin in yeast.

Patent publication EP196056 B1 (Chiron Corp.,) describes hSOD-proinsulin protein produced by yeast. The hSOD-proinsulin protein was subjected to cyanogen bromide cleavage and sulfite lysis prior to folding.

Hoechst described in EPO 379162 that "misrecombination of insulin precursors" can be

(i.e., recombinant insulin products with incorrect or partially incorrect intermolecular disulfide bonds) not by sulfite hydrolysis, but in an aqueous medium by combining the incorrect recombinant with Excess thiols react to convert to the correct insulin product. After the cleavage (chemical or enzymatic) of amino acids or peptide groups from the fusion polypeptide (which occurs after host cell lysis), the original sulfitololysis step is performed because the six cysteines of the insulin precursor are then amino acid into its S-sulfonate. In the subsequent renaturation step, native proinsulin is produced from the proinsulin S-sulfonate by formation of the correct three disulfide bonds. During this renaturation step, so-called "wrong recombinants" are produced.

Hoechst also discloses in PCT WO 91/03550 a method for preparing a "balanced composition" containing the desired protein (such as proinsulin). Sulphite solution is accomplished before folding, and after folding, the "equilibrium component" is cleaved simultaneously with the C-chain of proinsulin.

Additionally, Hoechst in EP 347781 B1 describes "small proinsulin" (B-Arg-A) and its use for pathogenic mono-Arg insulins and insulins. They also describe a fusion protein containing B-Arg-A and an "equilibrium component". The "equilibrium component" is cleaved with cyanogen bromide and sulfite lysed prior to folding of the polypeptide.

The present invention discloses an improved and efficient method for producing recombinant human insulin. A recombinant proinsulin hybrid polypeptide containing the leader sequence was synthesized in E. coli. After partial purification, it is folded, but with the leader peptide still attached, under conditions that allow the previous folding to take place. Biologically active human insulin is then produced by combined treatment with trypsin and carboxypeptidase B, where these enzymes cleave off both the leader peptide and the C chain. The purified human insulin produced is thus identical to naturally occurring human insulin.

The detrimental and cumbersome processes involved in CNBr cleavage of hybrid polypeptides and sulfite cleavage for protection of abundant SH groups are removed in the new method because the complete proinsulin hybrid polypeptide can be efficiently folded into its Native structure, even in the presence of a leader peptide and unprotected cysteine. Active recombinant human insulin is released by enzymatic cleavage, which is then purified.

Brief description of the drawings

In the restriction maps of the three plasmids listed in Figures 3-5, not all restriction sites on these plasmids were identified. However, those restriction sites necessary for a full understanding of the invention are listed.

Figure 1: Folded, disulfide-bonded proinsulin hybrid polypeptide produced by enzymatic cleavage of expression plasmid pBAST-R, resulting in human insulin. Only part of the SOD leader sequence is shown.

Figure 2: Folded, disulfide bonded proinsulin hybrid polypeptide produced by enzymatic cleavage of expression plasmid pBAST-LAT or plasmid pλBAST-LAT to yield human insulin. Only part of the SOD leader sequence is shown.

Figure 3: The structure of plasmid pBAST-R, an expression plasmid encoding SOD-proinsulin hybrid polypeptide, with the preservation number ATCC 69362.

Figure 4: The structure of plasmid pDBAST-LAT, an expression plasmid encoding SOD-proinsulin hybrid polypeptide, with the preservation number ATCC 69361.

Figure 5: The structure of plasmid pλBAST-LAT, an expression plasmid encoding SOD-proinsulin hybrid polypeptide, with the preservation number ATCC 69363.

Figure 6: Amino acid sequence and corresponding DNA nucleotide sequence of SOD-proinsulin hybrid polypeptide expressed with plasmid pBAST-R.

Figure 7: Amino acid sequence and corresponding DNA nucleotide sequence of SOD-proinsulin hybrid polypeptide expressed by plasmids pDBAST-LAT and pλBAST-LAT.

Figure 8: Human insulin production from proinsulin hybrid polypeptide expressed with plasmid pDBAST-LAT as a function of folding mixture pH.

Folding of the proinsulin hybrid polypeptide (produced as described in Example 2) was accomplished at 4°C for 16 hours with 1 mg/ml or 0.5 mg/ml hybrid polypeptide in 100 mM glycerol at various pHs. The folded material was treated with (trypsin 1:500w/w) (Sigma) and carboxypeptidase B (CPB, Sigma, 1:200w/w) at 37°C, pH9 for 30 minutes, and then treated by ¹²⁵ I-insulin (Amersham) Immunoreactivity (IR) was detected by standard radioimmunoassay with human recombinant insulin (Calbiochem).

Figure 9: Production of human insulin with proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT

The proinsulin hybrid polypeptide (produced as described in Example 2) was dissolved in 8 M urea, 5 mM HCl at a concentration of about 30 mg/ml, and then diluted to 1 mg/ml, pH 11.0, with 100 mM glycerol-NaOH. Fold at 22°C (room temperature) for 20 hours. The pH was then adjusted to 8.8 with HCl. Carboxypeptidase B (1:1000 w/w, Sigma) and trypsin (1:2000 w/w, Sigma) were added and the reaction mixture was incubated at 37°C for 60 minutes. The digestion mixture was acidified to pH 3 before dilution with 10 mM HCl. 150 μl of samples were analyzed by reverse-phase high-pressure liquid chromatography (RP-HPLC) on a 250×4 mm, 5 μLichrosphere 100 RP-8 column (Merch) with 50 mM tetraethylamine phosphate containing 31.5% (v/v) acetonitrile , 162mM Nacl ₄ , pH 3 equilibrated. The column was developed with a phenomenological gradient of 31.5%-40.5% acetonitrile over 75 minutes at a flow rate of 1 ml/min. Absorbance at 220 nm was detected.

A: 5ug standard insulin (Boehringer-Mannheim);

B: recombinant human insulin produced after enzymatic treatment;

C: Folded SOD-proinsulin hybrid polypeptide.

Figure 10: Proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT produces human insulin as a function of pH in the folding mixture

Proinsulin hybrid polypeptides (produced as described in Example 2) were diluted to 1 mg/ml with 100 mM glycerol-NaOH buffer at the indicated pH, and folded at 22°C for 16 hours. Enzyme treatment and RP-HPLC analysis were performed as described in FIG. 9 . The amount of recombinant human insulin produced from the hybrid polypeptide was calculated from the area of the peak having the same retention time as standard insulin.

Figure 11: Proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT produces human insulin as a function of ascorbic acid concentration in the refolding mixture

The SOD-proinsulin hybrid polypeptide (produced as described in Example 2) was folded at 1 mg/ml at 22°C in 100 mM glycerol-NaOH, pH 11.2, in the presence of the indicated concentrations of ascorbic acid. After a 5 and 25 hour folding process, the samples were treated with trypsin and carboxypeptidase B (according to Figure 9). The recombinant human insulin product was analyzed on RP-HPLC (according to Figure 9).

Figure 12: Reliability of human insulin produced from proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT

Fold the SOD-proinsulin hybrid polypeptide at 1 mg/ml in 100 mM glycerol-NaOH, pH 11.2 and 1.2 mM ascorbic acid at 22°C for 16 hours at 22°C. After enzyme treatment (according to Figure 9), the mixture was analyzed by chromatography on a DEAE-Sepharose column equilibrated at pH 8 with 20 mM Tris-HCl. Recombinant human insulin was eluted with a gradient of 0-0.4M NaCl in 20mM Tris-HCl, pH 8. The peak fractions were collected and acidified to pH 3 with HCl. As described in Figure 9, recombinant human insulin was further purified from insulin-like molecules by RP-HPLC. The main peak was collected, desalted on a Sephadex G-25 column with 0.25M acetic acid, and then lyophilized. Samples (5 ug recombinant human insulin) prepared in 10 mM HCl were analyzed by RP-HPLC under the same conditions.

A: standard insulin;

B: recombinant human insulin purified by HPLC;

C: Combined sample of HPLC purified recombinant human insulin and standard insulin.

Figure 13: Human insulin produced from proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT as a function of protein concentration

SOD-proinsulin hybrid polypeptides (produced as described in Example 2) were folded in 100 mM glycerol-NaOH, pH 11.2, at the indicated final protein concentrations of 0.5 mg/ml-10 mg/ml. Each folding mixture was supplemented with 2.5 mol of ascorbic acid per mol of SH group. Fold at 24°C (room temperature) for 16 hours. Enzymatic treatment and RP-HPLC analysis were performed as described in FIG. 9 .

Figure 14: Human insulin produced from crude intracellular precipitated proinsulin hybrid polypeptide expressed by plasmid pDBAST-LAT as a function of folding time

The intracellular pellet was dissolved in 20 mM glycerol-NaOH, 33 uM EDTA, pH 11.2 at a concentration of approximately 2.6 A ₂₈₀ per ml. The pH was adjusted to 12 with 10N sodium hydroxide. The solution was left and stirred for 10 minutes. The pH was brought dropwise to 11.2 with concentrated hydrochloric acid. Activated charcoal (acid washed, Sigma) was added to a final concentration of 0.1% w/v and the mixture was stirred for 30 minutes. The _A280 of the clarified supernatant was about 2.15. Ascorbic acid was supplemented to achieve a final concentration of 3 mM. With vigorous stirring at room temperature (22-23°C), the proinsulin hybrid polypeptide was folded as shown. At different time points of the test (starting from dissolving), 10ml samples were taken successively, dropped to pH 8.8, and then in the presence of 50uM ZnCl ₂ , with carboxypeptidase B (1:1000w/w) and trypsin (1: 2000w/w) digestion. Insulin content in each digested sample was determined by RP-HPLC analysis as described in FIG. 9 . The progression of the folding reaction was determined by the increase in insulin (after digestion) and the decrease in the level of free SH groups, the latter being detected by the Ellman reaction (16).

SUMMARY OF THE INVENTION

The present invention provides a method for producing human insulin, comprising folding a proinsulin-containing hybrid polypeptide under conditions that allow correct disulfide bond formation, enzymatically cleaving the folded, disulfide-bonded hybrid polypeptide to produce active human insulin, Active human insulin is then purified.

The present invention also provides a polypeptide comprising proinsulin and a leader peptide linked to the N-terminus of said proinsulin, wherein said polypeptide is folded and contains correct disulfide bonds.

Detailed description of the invention

The plasmids pBAST-R, pDBAST-LAT, pλBAST-LAT in Escherichia coli were deposited with American Type Culture on July 26, 1993, in accordance with and in accordance with the requirements of the Budapest Treaty for International Recognition of the Deposit of Microorganisms for Plasmid Purposes The Collection Center (ATCC) (12301 Parklawn Drive, Rochville, Maryland 20852) has accession numbers 69362, 69361 and 69363, respectively.

As used herein, a hybrid polypeptide contains a leader peptide covalently linked to the desired polypeptide. The hybrid polypeptide of the present invention contains proinsulin, preferably SOD as a leader peptide.

As used herein, folding includes folding hybrid polypeptides containing proinsulin that has not been cleaved with CNBr prior to folding and that has not undergone sulfite cleavage to protect the SH group prior to folding, wherein folding is such that correct disulfide bond formation in the hybrid polypeptide.

As used herein, the correct disulfide bond formation of the hybrid polypeptide includes the formation of three disulfide bonds between ^CysB7 - ^CysA7 , ^CysB19 - ^CysA20 and ^CysA6 - ^CysA11 of the insulin (labeled according to the numbering in the mature insulin Cys residues).

As used herein, proinsulin comprises a polypeptide comprising the B, C and A chains of insulin, from the N-terminus to the C-terminus.

As used herein, the C-chain peptide of insulin contains the naturally occurring C-peptide and any other oligopeptide, dipeptide or single amino acid that can be cleaved by trypsin and carboxypeptidase B.

As used herein, a leader peptide comprises any peptide or polypeptide covalently linked to the B chain of insulin which allows folding and disulfide bond formation and which is cleavable by trypsin. The leader peptide is preferably SOD.

As used herein, SOD includes the amino acid sequence of CuZnSOD or MnSOD of any substantial portion, and said portion does not have to have the biological activity of SOD, and compared with the amino acid sequence of naturally occurring SOD, said portion does not have to have the same amino acid sequence. DNA encoding SOD can be mutated using methods known in the art, such as Bauer et al. (1985), Gene, 37:73-81.

The leader peptide may include any other peptide, polypeptide or protein or the amino acid sequence of any substantial part of any of said peptides, polypeptides or proteins, except for SOD, wherein said part does not necessarily have biological activity as a peptide, polypeptide or protein, The moiety also need not have the same amino acid sequence as a naturally occurring peptide, polypeptide or protein; however, the leader peptide must allow the hybrid polypeptide to fold and form proper disulfide bonds.

As used herein, insulin may include homologues of natural insulin.

As used herein, proinsulin may include homologues of native proinsulin.

As used herein, the term "homolog" in relation to an insulin polypeptide produced by the method of the present invention refers to a polypeptide having substantially the same amino acid sequence and substantially the same biological activity as insulin. Thus, homologues may differ from insulin polypeptides produced by the methods of the invention by the addition, deletion or substitution of one or more non-essential amino acid residues, so long as the resulting polypeptide retains the biological activity of insulin. Those skilled in the art use well-known methods, including, for example, conventional methods for designing and preparing DNA sequences for bacterial expression of polypeptide homologues of polypeptides, modifying cDNA and genomic sequences by site-directed mutagenesis techniques, and constructing recombinant proteins And expression vectors, bacterial expression of polypeptides and routine biochemical methods to detect the biochemical activity of polypeptides, it is easy to determine which amino acid can be added, deleted or substituted (including which amino acid can be substituted).

The above definition of insulin homologues applies equally to the homologues of proinsulin.

Examples of insulin homologues produced by the method of the present invention are deletion homologues containing less than all residues of native insulin, substitution homologues in which one or more specific residues are replaced by other residues, and substitution homologues in which one or more amino acids Addition of residues to the terminal or middle portion of said insulin polypeptide, all of which have the biological activity of insulin.

Examples of homologues are the analogues disclosed in EPO patent application EP384472 and Eli Lilly's insulin analogue "Humalog" disclosed in "Eli Lilly and Company Report to Shareholder 1992".

Substantially identical amino acid sequences are defined herein as including substitutions and/or deletions and/or additions in the amino acid sequence, and according to, for example, Albert L. Lehninger, Biochemistry, 2nd edition, Worth Publishers Inc. (1975), 4 Chapter; Creighton, Protein Structure, Practical Methods, IPL Press at Oxford University Press, Oxford, England (1989); and Margaret O. Dayhoff, Atlas of Protein Sequence and Structure, Vol.5, The National Biomedical Research Foundation (1972), Chapter 9 Description Homologous or equivalent groups of , may include up to 10 residues. Such substitutions are well known to those skilled in the art.

The DNA encoding the insulin polypeptide can be mutated using methods well known to those skilled in the art, eg Bauer et al., (1985), Gene 37: 73-81. As described herein, the mutated sequence can be inserted into a suitable expression vector, which is introduced into cells which are then treated so that the mutated DNA expresses a polypeptide homologue.

The plasmid of the present invention containing the sequence encoding the proinsulin-containing hybrid polypeptide can be expressed in bacteria, yeast, fungi or mammalian cells such as CHO, chicken embryos, fibroblasts or other known cell lines, which usually also contain the Regulatory elements necessary for expression of the cloned gene in bacterial, yeast, fungal or mammalian cells are positioned with the nucleic acid encoding the hybrid polypeptide such that expression occurs. Regulatory elements required for expression include a promoter sequence that binds the RAN polymerase and a ribosome binding site that binds the ribosome.

The plasmid of the present invention expresses a proinsulin-containing hybrid polypeptide.

Those skilled in the art will appreciate that the plasmids deposited with respect to the present application can be easily altered to express homologous polypeptides using known techniques (eg, by site-directed mutagenesis or insertion of linkers). The technique is described, for example, in Sambrook J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press.

Appropriate regulatory elements are located within the plasmid associated with the DNA encoding the proinsulin-containing polypeptide in order to effect expression of the hybrid polypeptide in a suitable host cell. In preferred embodiments of the invention, regulatory elements are located near and upstream of the DNA encoding the hybrid polypeptide.

The present invention also includes various ribosome binding sites (RBS) for providing mRNA transcribed from DNA encoding a proinsulin-containing hybrid polypeptide for binding to ribosomes in the host cell, such as deo RBS.

The plasmids of the present invention also contain an ATG initiation codon. The DNA encoding the proinsulin-containing hybrid polypeptide is in phase with the ATG initiation codon.

The plasmids of the present invention also include DNA sequences containing replication origins derived from bacterial plasmids capable of autonomous replication in host cells. A suitable origin of replication can be obtained from various sources, such as from plasmid pBR322 (ATCC No. 37016).

The plasmids of the present invention comprise DNA sequences comprising genes associated with selectable or identifiable phenotypic characteristics, such as drug resistance genes, such as ampicillin resistance, chloramphenicol or penicillin resistance, when the plasmid is present in the host, The phenotypic features will then manifest.

Examples of vectors that can be used to express a hybrid polypeptide (including proinsulin) are viruses, such as bacterial viruses, such as bacteriophages (such as lambda phage), cosmids, plasmids and other vectors. The gene encoding the proinsulin-containing hybrid polypeptide is inserted into an appropriate vector by methods well known in the art. The insert and vector DNA can be cleaved, for example using conventional restriction endonuclease sites, to generate splice-paired complementary ends, which are then ligated together using DNA ligase. Alternatively, a synthetic linker containing a base sequence complementary to a restriction site in the vector DNA can be ligated to the insert DNA, followed by digestion with a restriction enzyme that cleaves at the given site. Other methods may also be employed.

A preferred bacterial host cell is an E. coli cell. Suitable E. coli cells are strains Sφ733 (cytRstrA) or 4300, but other E. coli strains and other bacteria can also be used as plasmid hosts.

The bacteria used as the host can be any strain, including auxotrophic (such as A1645), prototrophic (such as A4255) and lytic strains; F ⁺ and F ^- strains; strains containing the cI857 repressor sequence of λ prophage (such as A1645 and A4255) and strains without the deo repressor and/or the deo gene (see European Patent Application Publication 030972, published February 22, 1989). Escherichia coli strain φ733 and Escherichia coli strain 4300 have been deposited at ATCC with deposit numbers 69361 and 69363, respectively.

All of the above E. coli host strains may be "cured" of the plasmids they carry, using methods well known in the art, as described by R.P Novick (Bacteriol. Review 33, 210 (1969)).

The present invention provides a method of producing insulin comprising folding a proinsulin-containing hybrid polypeptide under conditions that allow formation of correct disulfide bonds, enzymatic cleavage of (processing) the folded, disulfide-bonded hybrid polypeptide to produce insulin , and then purify the insulin. The insulin has the activity and properties of commercially available human insulin.

In a preferred embodiment, folding comprises incubating the hybrid polypeptide at about pH 8.5-12.0 at 4-37°C for about 1-30 hours.

In another preferred embodiment, folding comprises incubating the hybrid polypeptide at about pH 8.5-12.0 in the presence of ascorbic acid for about 1-30 hours at 4-37°C.

In a particularly preferred embodiment, the pH during folding is between 11.0 and 11.25.

In another particularly preferred embodiment, the concentration of ascorbic acid in the folding mixture is about 2 moles of ascorbic acid per mole of SH groups.

In another embodiment, the incubation time is about 5 hours.

In another embodiment, the treatment comprises adjusting the pH to about 8.8-9.0, followed by cleavage of the hybrid polypeptide with trypsin and carboxypeptidase B at 16-37°C for about 30 minutes to 16 hours.

In another embodiment, purification comprises DEAE-Sepharose chromatography and RP-HPLC.

In another embodiment, purification also includes ultrafiltration and CM-Sepharose chromatography.

In a particularly preferred embodiment, the purification also includes DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography.

In a particularly preferred embodiment, the hybrid polypeptide is expressed using the plasmid pDBAST-LAT deposited with the ATCC under Accession No. 69361.

In a preferred embodiment, the hybrid polypeptide is expressed using the plasmid pλDBAST-LAT deposited with the ATCC under Accession No. 69363.

In a preferred embodiment, the hybrid polypeptide is expressed using the plasmid pBAST-LAT deposited with the ATCC under Accession No. 69362.

In a preferred embodiment, the hybrid polypeptide is obtained by treating bacterial cells containing DNA encoding the hybrid polypeptide so that the DNA directs its expression and then recovering the hybrid polypeptide from said cells.

It is contemplated that the treatment involves fermentation in the presence of glucose, glycerol or galactose.

It is further contemplated that the recovery of the hybrid polypeptide from the cell involves disrupting the cell wall of the bacterial cell or fragments thereof to produce a lysate, separating the intracellular precipitate from the lysate by centrifugation, lysing the precipitate, and optionally purifying the hybrid by chromatography or ultrafiltration peptide.

The present invention also provides a polypeptide comprising proinsulin and a leader peptide attached to the N-terminus of said proinsulin, wherein said polypeptide is folded and contains correct disulfide bonds.

In a preferred embodiment, the leader peptide is derived from the N-terminus of CuZnSOD.

In a particularly preferred embodiment, the leader peptide contains 62 amino acids preceded by Met and followed by Arg residues.

In a preferred embodiment, the proinsulin comprises an insulin B-chain linked to an insulin A chain via a single Arg residue.

In another embodiment, the proinsulin comprises an insulin B chain linked to an insulin A chain via the dipeptide Lys-Arg.

The two proinsulin molecules are produced as hybrid proteins, otherwise the expression levels are very low and not commercially significant.

In all preferred embodiments, the Cys residues of the leader peptide are replaced with Ser residues.

Example

The following examples are provided to facilitate the understanding of the present invention, but are not intended and should not limit its scope in any way. The examples do not include detailed descriptions of routine methods used to construct vectors, insert genes encoding the polypeptides into said vectors, or introduce resulting plasmids into hosts. The examples also do not include detailed descriptions of routine methods used to detect polypeptides produced using the host-vector system. Such methods are well known to those skilled in the art and are described in a large literature including the following examples:

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

Example 1

Construction of production plasmids pBAST-R, pBAST-LAT and pλBAST-LAT expressing SOD-proinsulin hybrid polypeptide

Bacterial expression vectors were constructed under the control of the deoP ₁ P ₂ or λ _PL promoters for the production of hybrid proteins in E. coli. Proinsulin was produced as a hybrid protein because bacteria containing an expression vector encoding insulin B-chain-Lys-Arg-insulin A-chain were found to produce no detectable polypeptide. The hybrid protein contains a 62 amino acid long leader peptide derived from the N-terminus of CuZnSOD (11), a Met residue before the N-terminus of the leader peptide, and an Arg residue after the C-terminus, which makes It is attached to the insulin B chain. The insulin B chain is linked to the insulin A chain via a short C-chain peptide consisting of Lys-Arg or Arg. The two Cys originally present in SOD were replaced by Ser residues.

A plasmid pBAST-R

A series of plasmids were constructed to obtain pBAST-R, which can guide the effective expression of proinsulin hybrid polypeptide for human insulin production after transformation of appropriate Escherichia coli host cells.

The structure of plasmid pBAST-R is listed in Fig. 3, and this plasmid encodes SOD-insulin B-chain-Lys-Arg-insulin A chain hybrid polypeptide; The DNA sequence of said hybrid polypeptide and corresponding amino acid sequence.

Plasmid pBAST-R is about 4380bp long and contains the following elements (in counterclockwise direction):

The 11521bp long DNA fragment spans the AatII-MscI site on pBR322, including the tetracycline resistance gene.

A 21497bp long DNA fragment spanning the scaI-HaeII site on pBR322, including the truncated ampicillin resistance gene and DNA replication source.

The 3930bp long DNA fragment spans the AvaII-PpuMI site in E. coli, including the deo P ₁ P ₂ promoter and ribosome binding site (RBS) (13).

A 4188bp DNA fragment spanning the NdrI-PpuMI site of human CuAnSOD cDNA. Cys at positions 6 and 57 of mature SOD were replaced with Ser by oligonucleotide-directed mutagenesis (12).

5172bp long synthetic DNA fragment with PpuMI and BamHI ends. This region encodes Arg-insulin B chain-Lys-Arg-insulin A chain.

6 Synthetic 36bp multiple cloning site polylinker with BamHI and HindIII ends.

7 Synthetic 44bp oligonucleotide containing TrpA transcription terminator with HindIII and AatII ends (10).

The plasmid pBAST-R, which confers tetracycline resistance and encodes a SOD-insulin B chain-Lys-Arg-insulin A chain hybrid polypeptide, was introduced into Escherichia coli strain Sφ733 (cytRstrA), and then deposited at ATCC on July 26, 1993, The deposit number is ATCC69362.

B Plasmid pDBAST-LAT

Another series of plasmids was constructed to obtain the plasmid pDBAST-LAT, which can direct the efficient and high-level expression of the proinsulin hybrid polypeptide for human insulin production after transformation of appropriate Escherichia coli host cells.

Figure 4 lists the structure of the plasmid pDBAST-LAT encoding the SOD-insulin B chain-Arg-insulin A chain hybrid polypeptide; Figure 7 lists the DNA sequence and corresponding amino acid sequence of the hybrid polypeptide. Plasmid pDBAST-LAT is about 4377bp long and contains the following elements (in counterclockwise direction):

1. A DNA fragment of 1521 bp spanning the AatII-MscI site on pBR322, including the tetracycline resistance gene.

2. A DNA fragment of 1497 bp spanning the ScaI-HaeII site on pBR322, including a truncated ampicillin resistance gene and a DNA replication source.

A 3.930bp long DNA fragment spanning the AvaII-PpuMI site on Escherichia coli, including the deo P ₁ P ₂ promoter and ribosome binding site (RBS) (13).

4. 188bp long DNA fragment spanning the NdrI-PpuMI site of human CuAnSOD cDNA. By oligonucleotide-directed mutagenesis (12), Ser was used to replace Cys at positions 6 and 57 of mature SOD, and the GC content of this fragment was reduced to 38%.

5.169bp long synthetic DNA fragment with PpuMI and BamHI ends. This region encodes Arg-insulin B chain-Lys-Arg-insulin A chain.

6. Synthetic 36bp multi-cloning site polylinker with BamHI and HindIII ends.

7. Synthetic 44bp oligonucleotide containing TrpA transcription terminator with HindIII and AatII ends (10).

The plasmid pDBAST-LAT, which confers tetracycline resistance and encodes a SOD-insulin B chain-Lys-Arg-insulin A chain hybrid polypeptide, was introduced into Escherichia coli strain Sφ733 (cytRstrA), and then deposited at ATCC on July 26, 1993, Deposit number is ATCC 69361.

C Plasmid pλBAST-LAT

Another series of plasmids was constructed to obtain plasmid pλBAST-LAT, which, after transformation of appropriate E. coli host cells (containing the cI1857 repressor), can direct the efficient and high-level expression of the proinsulin hybrid polypeptide for human insulin production.

Figure 5 shows the structure of the plasmid pλBAST-LAT encoding the SOD-insulin B chain-Arg-insulin A chain hybrid polypeptide. The DNA sequence and corresponding amino acid sequence of the hybrid polypeptide are listed in FIG. 7 .

Plasmid pλBAST-LAT is about 3777bp long and contains the following elements (in counterclockwise direction):

1. A 1521bp DNA fragment spanning the AatII-MscI site on pBR322, including the tetracycline resistance gene.

2. A 1497bp DNA fragment spanning the ScaI-HaeII site on pBR322, including the truncated ampicillin resistance gene and DNA replication source.

3. The 330bp long DNA fragment spans the BamHI-EcoRI site on the plasmid pSOFα13(14), including the _λPL promoter and an AvrII-NdeI 30 base pair long deo ribosome binding site.

4. 188bp long DNA fragment spanning the NdrI-PpuMI site of human CuAnSOD cDNA. By oligonucleotide-directed mutagenesis (12), Ser was used to replace Cys at positions 6 and 57 of mature SOD, and the GC content of this fragment was reduced to 38%.

5. 169bp long synthetic DNA fragment with PpuMI and BamHI ends. This region encodes Arg-insulin B chain-Lys-Arg-insulin A chain.

6. Synthetic 36bp multi-cloning site polylinker with BamHI and HindIII ends.

7. Synthetic 44bp oligonucleotide containing TrpA transcription terminator with HindIII and AatII ends (10).

The plasmid pλBAST-LAT, which confers tetracycline resistance and encodes a hybrid polypeptide of SOD-insulin B chain-Lys-Arg-insulin A chain, was introduced into Escherichia coli strain 4300 (F-, bio, cI ⁸⁵⁷ ), and then in July 1993 It was deposited at ATCC on the 26th with the accession number ATCC 69363.

Propagate bacterial cells at 30°C. After the temperature was raised to 42°C, the production of the hybrid polypeptide was induced.

Example 2

Fermentation, growth conditions and purification of SOD-proinsulin hybrid polypeptide

I stock culture

Make the former culture of the escherichia coli bacterial strain Sφ733 that contains plasmid pDBAST-LAT (or pBAST-R) in the casein medium (20gr/L casein hydrolyzate, 10gr/yeast extract and 5gr/LNaCl). The cultures were then diluted two-fold with freezing medium and stored at -80°C.

Freezing medium:

K ₂ HPO ₄ 6.3gr

KH ₂ PO ₄ 1.8gr

Sodium citrate 0.45gr

MgSO ₄ 7H ₂ O 0.09gr

(NH ₄ ) ₂ SO ₄ 0.9gr

Glycerin 44gr

Each 500ml

II inoculum

The inoculum was grown in production medium (see below). Sterilized media in shaker flasks were inoculated from the original culture and incubated at 37°C, 200 rpm for 15 hours. Subsequent stages of inoculum propagation were accomplished, if desired, in stirred, aerated fermenters. Inoculate the sterile medium with 2-10% flask culture, then incubate for 15 hours at 37°C, pH 7±0.5, while stirring and aerating to maintain the dissolved oxygen level above 20% air saturation.

III production

Production medium:

K ₂ HPO ₄ 8gr/L

KH ₂ PO ₄ 2gr/L

Sodium citrate 2gr/L

NH ₄ Cl 3gr/L

K ₂ SO ₄ 0.6gr/L

FeSO ₄ ·7H ₂ O 0.04gr/L

MgSO ₄ 7H ₂ O 0.4gr/L

CaCl ₂ 2H ₂ O 0.02gr/L

Trace element solution 3ml/L

Tetracycline 0.01gr/L

Glucose 2gr/L

Glycerin 1ml/L

Trace element solutions:

MnSO ₄ ·H ₂ O 1gr/L

ZnSO ₄ 7H ₂ O 2.78gr/L

CoCl ₂ 7H ₂ O 2gr/L

Na ₂ MoO ₄ ·2H ₂ O 2gr/L

CaCl ₂ 2H ₂ O 3gr/L

CuSO ₄ 5H ₂ O 1.85gr/L

H ₃ BO ₃ 0.5gr/L

HCl(32%) 100ml/L

The production medium was inoculated with 0.5-10% of the inoculum culture and then cultured at 37°C. The agitation-aeration rate was set so that the dissolved oxygen level was maintained at about 20% of the air saturation. The pH was maintained at 7 ± 0.2 with _NH3 .

Enter a sterile solution of 50% dextrose and 30% glycerol to supplement energy and carbon sources. When the cell concentration reached an _OD660 of 25, a sterilized solution of 10% glucose and 30% glycerol was infused, and then continued to grow for about 5 hours until the cell concentration reached an _OD660 of approximately 60. The culture was then quenched and the cells recovered by centrifugation. In the presence of any one of glucose, glycerol, galactose or a combination thereof as a carbon source, it is beneficial to express the SOD-proinsulin hybrid polypeptide against Escherichia coli.

IV purification

The SOD-proinsulin hybrid polypeptide expressed by the plasmids pBAST-R and pDBAST-LAT accumulated in the intracellular precipitate, which was separated by the following method: the bacterial cake of 1 gr (net weight) was suspended in 10 ml containing 50 mM Tris-HCl , pH 8, in 10mM EDTA buffer, treated with lysozyme (Merck, 2500u/ml) for 2 hours at 37°C. The mixture was then sonicated, and Nonidet-P-40 (Sigma) or Triton X 100 was added to a final concentration of 2%, followed by stirring at room temperature for 2 hours. The precipitate was pelleted by centrifugation and washed with water.

The hybrid polypeptide was purified to near homogeneity by anion exchange chromatography as follows. The precipitate was dissolved in 8M urea, 20 mM Tris-HCl, 200 mM β-mercaptoethanol, pH 8.2. The solution was clarified by centrifugation and then chromatographed on a 20DEAE-Sepharose Fast-Flow column (Pharmacia LKB) pre-equilibrated with 8M urea Tris-HCl, 200 mM β-mercaptoethanol, pH 8.2. The effluent was collected and the hybrid protein was precipitated with 40% saturation (NH ₄ )SO ₄ or concentrated by ultrafiltration on a 10K membrane followed by diafiltration with 100 mM Glycine-HCl, pH 3.1.

In addition, the SOD-proinsulin hybrid polypeptide expressed by plasmid pBAST-R was purified to uniformity by dissolving in 8M urea, 20mM dithiothreitol, 50mM sodium acetate, pH5, and then passing through 100kD and 50kD membrane (Filtron) series membranes. . Hybrid polypeptides were concentrated on 10 kD membranes and precipitated with ( _NH4 ) _SO4 at 40% saturation.

Example 3

Folding and enzymatic cleavage of SOD-proinsulin hybrid polypeptide

The proinsulin hybrid polypeptide obtained by (NH ₄ )SO ₄ precipitation or ultrafiltration (Example 2) was dissolved in 8M urea, 5mM Hcl, and then diluted into 100mM glycerol buffer, pH8.5-12.0, the final concentration is about 1 mg/ml.

A Fold the SOD-proinsulin hybrid polypeptide expressed by plasmid pBAST-R in about 1-24 hours at about 4-37°C so as to form correct disulfide bonds.

The pH of the solution containing the folded disulfide-bonded hybrid polypeptide is adjusted to about 8.8-9.0 with HCl, and the protein is treated with trypsin and carboxypeptidase B at 16-37°C for 30-120 minutes.

After many experiments, it was found that the optimal conditions were as follows: the hybrid polypeptide expressed by plasmid pBAST-R was dissolved in 8M urea, 5mM HCl, then diluted to 100mM glycerol buffer, pH11.0 (Figure 8), and the final concentration was about 1mg/ ml, thereafter, the hybrid polypeptide was folded at 25°C for 6-16 hours, and thereafter, at 37°C, the folded disulfide The bonded hybrid polypeptide is cleaved for 30-60 minutes.

Figure 1 schematically illustrates the enzymatic cleavage of the folded disulfide-bonded proinsulin hybrid polypeptide expressed from plasmid pBAST-R to produce insulin.

B Fold the SOD-proinsulin hybrid polypeptide expressed by plasmid pBAST-LAT in about 5-30 hours at about 7-31° C. so as to form correct disulfide bonds.

The pH of the solution containing the folded disulfide-bonded hybrid polypeptide was adjusted to about 8.8-9.0 with HCl, and the protein was treated with trypsin and carboxypeptidase B for 16 hours at 22-37°C.

After many experiments, it was found that the optimal conditions were as follows: the hybrid polypeptide expressed by plasmid pBAST-LAT was dissolved in 8M urea, 5mM HCl, then diluted to 100mM glycerol buffer, pH11.0-11.25 (Figure 8), and the final concentration was about 1mg/ml, thereafter, fold the hybrid polypeptide at 25°C for 6-16 hours, and thereafter, at 25°C, use trypsin (1:15.00w/w) and carboxypeptidase B (1:10.00w/w) to The folded disulfide-bonded hybrid polypeptide was cleaved for 16 hours.

Figure 2 schematically illustrates the enzymatic cleavage of the folded disulfide-bonded proinsulin hybrid polypeptide expressed from plasmid pBAST-LAT to produce insulin.

Specific conditions for A and B above are specified in the legends to Figures 8-14.

Example 4

Protein Analysis and Purification of Human Insulin from SOD-Proinsulin Hybrid Polypeptide Expressed from Plasmid pBAST-R

Using commercially available human insulin as a standard (Calbiochem), the human insulin obtained from the SOD-proinsulin hybrid polypeptide expressed from plasmid pBAST-R was determined by radioimmunoassay and RP-HPLC. The theoretical yield of recombinant human insulin calculated based on the amino acid sequence of proinsulin was 45.6%. As can be seen from Figure 8, the optimal folding is at pH11. At this pH, insulin was produced at about 80% of theoretical yield (equivalent to about 40% of the input hybrid polypeptide). Insulin produced from the proinsulin hybrid polypeptide expressed from plasmid pBAST-R was detected by RP-HPLC. Use Vydac 218TP54, 250×4.6mm I.D (Separationgroup), 5um, 300A pore size column at room temperature, with a flow rate of 1ml/min. As eluent A, 0.1% trifluoroacetic acid (TFA) in water was used. 0.08% TFA in acetonitrile was used as eluent B. The column was washed with equilibration buffer (25% eluent B) for 5 minutes, followed by a linear gradient of 25-50% eluent B over 37.5 minutes. Absorbance at 220nm or 280nm is detected. Analysis of human insulin following enzymatic digestion of the folded, disulfide-bonded hybrid polypeptide by reversed-phase high-pressure liquid chromatography revealed a major peak with the same retention time as standard human insulin.

Two small batches were prepared to yield 26 mgh and 13 mg of human insulin respectively. Human insulin was purified from the enzyme-treated solution (pH 9) by ultrafiltration on a 3K or 5K membrane (Filtron), followed by CM-Sepharose chromatography (citrate buffer, pH 3). Peak fractions were desalted, lyophilized, and subjected to N-terminal sequencing and amino acid analysis. The amino acid composition of these two batches of recombinant human insulin was essentially the same as that of native human insulin (see Table 1, Formulation 1). The amino-terminal 5 amino acid sequences of insulin preparations were determined by Edamn degradation method. The same was found for the _NH2 -termini of the A and B chains of human insulin, confirming the reliability of the in vitro product.

However, sequencing results indicated that about 25% of the molecules had an additional Arg in the first position. This result is consistent with trypsin cleavage between Lys and Arg (within the linker sequence Lys-Arg), leaving an Arg residue at the amino terminus of the A chain.

It was found that completion of the reaction at pH 11 can be used to specifically hydrolyze the C-terminus of Arg with trypsin. At this elevated pH, most of the ε-amino groups of Lys are uncharged (pK=10.3), thus enabling selective cleavage. By completing the trypsin step at pH 11 (see Table 1, formulation 2), followed by digestion with carboxypeptidase B at pH 8.5, these two batches yielded 1 mg and 6.5 mg of purified insulin, respectively. N-terminal sequencing showed that the amount of insulin containing extra Arg was reduced to about 5%.

Table 1

Amino acid composition of recombinant human insulin amino acid number of residues theoretical value standard insulin Preparation 1 Preparation 2 Asx 3 3.20 3.38 3.26 Thr 3 2.98 2.83 2.68 Ser 3 2.84 2.53 2.77 Glx 7 7.15 7.73 7.23 Pro 1 1.28 1.13 1.09 Gly 4 4.24 4.39 4.25 Ala 1 1.00 1.28 1.04 Cys 6 5.88 5.11 5.79 Val 4 3.82 4.58 3.88 Ile 2 2.04 1.96 1.96 Leu 6 5.87 6.10 5.99 Tyr 4 3.80 3.80 3.87 Phe 3 3.15 3.56 3.03 His 2 2.04 2.05 2.08 Lys 1 1.01 1.05 1.02 Arg 1 0.96 1.30 1.18

Formulations 1 and 2 represent the amino acid composition of recombinant human insulin produced from the proinsulin hybrid polypeptide expressed from plasmid pBAST-R. Trypsin cleavage was done at pH 9 (Preparation 1) or pH 11 (Preparation 2).

Amino acid analysis was performed after performic acid oxidation and gas-phase hydrolysis of purified insulin preparations.

Example 5

Peptide Analysis of Purified Human Insulin Produced from SOD-Proinsulin Hybrid Polypeptide Expressed from Plasmid pBAST-R

The purified human insulin produced according to the above example was subjected to peptide analysis using the endoprotease Glu-C (Sigma), which hydrolyzes the peptide bond at the carboxyl side of the glutamyl group.

In more detail, insulin samples expressed by cleaving the folded, disulfide bonds of the plasmid pBAST-R were digested with 5 ug Glu-C in 100 ul 0.1 M Tris-Hcl, pH 7.8, for 6 hours at 37°C. produced from a hybrid proinsulin polypeptide. Complete HPLC analysis: samples of commercial (control) insulin and insulin produced by cleavage of the folded, disulfide-bonded proinsulin hybrid polypeptide expressed from plasmid pBAST-R were acidified to about pH 3 and separated by RP-HPLC . Use Vydac 218TP54, 250×4.6mm ID (Separation group), 5um, 300A pore size column. The column was equilibrated with 31.5% (v/v) acetonitrile in 50 mM tetraethylamine phosphate, 162 mM NaClO ₄ , pH 3, and then developed with a linear gradient of 35-45% acetonitrile over 75 minutes at a flow rate of 1 ml/min. color. Absorbance was measured at 220 nm.

Following the control reaction all expected peptides were produced, even a small shoulder corresponding to one of the fragments after the peak possibly corresponding to the des-Thr( _B30 ) insulin-like molecule (15).

Examples 4 and 5 demonstrate that the recombinant polypeptide expressed from plasmid pBAST-R contains native human insulin sequences. A small fraction of recombinant proteins produced include Arg (Ao), desamido- or des-Thr (B ₃₀ ) insulin-like molecular forms. These unwanted by-products can be removed by chromatographic methods, such as RP-HPLC as described above.

Example 6

Protein Analysis and Purification of Human Insulin from SOD-Proinsulin Hybrid Polypeptide Expressed from Plasmid pBAST-R

In order to avoid the production of Arg (Ao) insulin by-products (Examples 4 and 5), the expression plasmid pBAST-R was modified to obtain DNA containing only the Arg residue between the AB chains of the hybrid polypeptide encoding proinsulin, which is different from that encoding Lys-Arg The DNA of Lys-Arg is located between the AB chains of the proinsulin hybrid polypeptide expressed by the plasmid pBAST-R. Expression plasmids pDBAST-LAT (Example 1B) and pλBAST-LAT (Example 1C) were obtained.

Insulin is efficiently produced following folding and enzymatic treatment with trypsin and CPB of the folded, disulfide-bonded proinsulin hybrid polypeptide expressed from the new expression plasmid pDBAST-LAT. Fewer insulin-like contaminants were present (Figure 9). Optimal folding was at pH 11.25 (Figure 10), and folding was significantly increased in the presence of about 2 moles of ascorbic acid per mole of SH groups in the reaction mixture (Figure 11).

Under other optimal folding conditions, a series of reactions was used to determine the effect of protein concentration on insulin production from proinsulin hybrid polypeptides. The yield was optimal when the protein concentration did not exceed 1.5 mg/ml (Figure 13).

Insulin was purified by DEAE-Sepharose chromatography followed by RP-HPLC (as described in Figure 9). As can be seen from Figure 12, the recombinant human insulin produced had the same residence time as standard (commercial) human insulin. The amino acid composition of the purified recombinant human insulin preparation was identical to that of standard insulin (see Table 2).

Table 2 shows that insulin produced from the proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT does not have the extra Arg attached to the insulin A chain (Arg(Ao)insulin) as described in Example 4. Therefore, the preferred plasmid for producing insulin is the plasmid pDBAST-LAT, and the preferred sequence of the proinsulin hybrid polypeptide is listed in FIG. 7 .

Table 2

Amino acid composition of recombinant human insulin amino acid number of residues theoretical value standard insulin recombinant insulin ABx 3 3.20 3.32 Thr 3 2.98 2.73 Ser 3 2.84 2.71 Glx 7 7.15 7.41 Pro 1 1.28 1.02 Gly 4 4.24 4.46 Ala 1 1.00 1.09 Cys 6 5.88 5.28 Val 4 3.82 4.00 Ile 2 2.04 1.91 Leu 6 5.87 6.34 Tyr 4 3.80 3.64 phe 3 3.15 3.06 His 2 2.04 2.18 Lys 1 1.01 1.02 Arg 1 0.96 1.07

The amino acid composition of standard human insulin and recombinant human insulin produced from proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT is shown.

Amino acid analysis was accomplished after performic acid oxidation and gas-phase hydrolysis of purified insulin preparations.

Example 7

Production of Human Insulin from the Proinsulin Hybrid Polypeptide Expressed from Plasmid pDBAST-LAT from Crude Intracellular Precipitate

An improved method for folding and converting the proinsulin hybrid polypeptide to insulin can be accomplished using the crude intracellular precipitate, omitting the initial purification step in Example 2, Part IV. Efficient production of insulin follows enzymatic cleavage of the folded, disulfide-bonded proinsulin hybrid polypeptide with trypsin and carboxypeptidase B (Figure 14 and Table 3). Insulin yield was calculated as the starting protein concentration (A ₂₈₀ ) determined for the precipitate solubilization step at pH 12 ( FIG. 14 ). It was shown that the SOD-proinsulin hybrid polypeptide was folded from the crude intracellular precipitate at about 4.5 hours from the start of the experiment ( FIG. 14 ).

Table 3 summarizes the partial purification of insulin from the proinsulin hybrid polypeptide expressed from plasmid pBAST-LAT from the crude intracellular pellet from one liter of fermentation culture plasmid at _OD660 =45. Dissolving and folding were accomplished as described in Figure 14. At 4.5 hours after initiation, the folded stack containing the folded, disulfide-bonded proinsulin hybrid polypeptide was solubilized and titrated to pH 8.8 with concentrated hydrochloric acid. Add _znCl2 (to a final concentration of 50uM), carboxypeptidase B (1:4000w/w) and trypsin (1:6000w/w). Digestion was performed at 37°C for 3 hours and then terminated by the addition of phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 0.5 mM. Analysis by HPLC (as described in Figure 9) showed a yield of 169 mg of insulin. Insulin is purified by sequential anion exchange and hydrophobic chromatography steps. The digested refolding mixture was loaded at approximately 50 A ₂₈₀ units per ml resin onto a DEAE Sepharose Fast Flow (Pharmacia) column pre-equilibrated with 20 mM Tris-HCl, 10 mM NaCl pH 8 buffer. Bound material was washed with 20 mM Tris-HCl, 100 mM NaCl pH 8 buffer and then eluted with 250 mM NaCl in the same buffer. The insulin-containing pool fraction represented 20% of the loaded protein with a purity of 37.1%. Ammonium sulfate was added to the DEAE elution pool to a concentration of 410 mM and then loaded at approximately ₂₈₀ units per ml of resin 12A onto a Phenyl-Sepharose Fast Flow column pre-equilibrated with 20 mM Tris HCl, 540 mM ammonium sulfate. Bound material was washed with equilibration buffer and insulin was eluted with 20 mM Tris HCl, 220 mM ammonium sulfate, pH 8 buffer. The fraction containing insulin represented 42.3% of the loaded protein with a purity of 74.1%. The result of this partial purification step was 120 mg (equivalent to standard insulin) produced with a yield of 5.16% insulin. Further purification can be performed by methods known in the art, such as gel filtration, RP-HPLC and crystallization (17).

table 3

Purification of recombinant human insulin produced from proinsulin hybrid polypeptide expressed from plasmid pDBAST-LAT after lysis of crude intracellular precipitate, folding and enzymatic treatment with trypsin and carboxypeptidase B Purification step A ₂₈₀ Minimal amount of insulin purified by HPLC %purity Precipitate dissolved 2326 - - Activated carbon treatment 1915 - - Folding and enzymatic processing 1915 169 8.8 DEAE-Sepharose pool 383 142 37.1 Phenyl Sepharose Pool 162 120 74.1

A ₂₈₀ represents the total absorbance at 280 nm for each purification step. The presence of insulin was determined by HPLC with reference to standard insulin as described in Figure 9 and corresponds to the main insulin peak of standard insulin.

references:

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8. Frank, B.H.and Chance, R.E.(1985), Preparation and characterization of recombinant DNA source of human insulin, used for therapeutic drugs produced by genetic engineering (The preparation and characterization of human insulin of recombinant DNA origin, in Therapeutic agents produced by genetic engineering,), Quo Vadissymposium, sanofi group, 29-30 May 1985, Toulouse-Labege, France, pp: 137-146.

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Claims (18)

1. A method for producing insulin, comprising: (a) obtaining a proinsulin-containing hybrid polypeptide by treating a bacterial cell containing DNA encoding the hybrid polypeptide to express and recover the hybrid polypeptide from the cell; (b) folding a proinsulin-containing hybrid polypeptide without sulfite hydrolysis of the hybrid polypeptide under conditions that allow the formation of correct disulfide bonds; (c) enzymatically cleaving the folded, disulfide-bonded hybrid polypeptide to produce insulin; (d) Purified insulin. 2. The method according to claim 1, wherein step (b) further comprises incubating the hybrid polypeptide. 3. The method according to claim 2, wherein the incubation is carried out at a temperature of 4-37°C. 4. The method according to claim 2, wherein the incubation is carried out for 1-30 hours. 5. The method according to claim 2, wherein the incubation is performed at pH 8.5-12.0. 6. The method according to claim 2, wherein the incubation is carried out in the presence of ascorbic acid. 7. The method according to claim 6, wherein the concentration of ascorbic acid in the folding mixture is 1-10 moles of ascorbic acid per mole of SH groups. 8. The method according to claim 5, wherein the pH is 11.0-11.25. 9. The method according to claim 6, wherein the pH is 11.0-11.25. 10. The method according to claim 1, wherein step (c) further comprises: (1) adjust the pH to 8.8-9.0; and (ii) Cleavage of the hybrid polypeptide with trypsin and carboxypeptidase B at 16-37°C for 30 minutes-16 hours. 11. The method according to claim 1, wherein step (d) further comprises purification with DEAE-Sepharose chromatography and RP-HPLC. 12. The method according to claim 1, wherein step (d) further comprises purification by ultrafiltration and CM-Sepharose chromatography. 13. The method according to claim 1, wherein step (d) further comprises using DEAE-Sepharose chromatography and phenyl-Sepharose chromatography. 14. The method according to claim 1, wherein the proinsulin hybrid polypeptide is expressed using plasmid pDBAST-LAT with ATCC Accession No. 69361. 15. The method according to claim 1, wherein the proinsulin hybrid polypeptide is expressed using the plasmid pλBAST-LAT with ATCC Accession No. 69363. 16. The method according to claim 1, wherein the proinsulin hybrid polypeptide is expressed using the plasmid pBAST-R with ATCC Accession No. 69362. 17. The method according to claim 1, wherein said treating comprises fermentation in the presence of glucose, glycerol or galactose. 18. The method of claim 1, wherein said recovering comprises: (1) disrupting the cell wall or fragments thereof of said bacterial cells to produce a lysate; (ii) separating the intracellular precipitate from the lysate by centrifugation; and (iii) Dissolving the precipitate.

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