RU2238951C2 - Chimeric protein providing formation of bioactive conformation, method for preparing protein with insulin activity, method for identification of peptidyl fragment - Google Patents

Abstract

FIELD: biotechnology, protein genetic engineering, proteins, biochemistry.

SUBSTANCE: invention can be used for preparing correctly folded chimeric protein comprising the insulin precursor. The chimeric protein comprises a peptidyl fragment with length 20-92 amino acids that shows 60% of identity with the first 20 N-amino acid residues SEQ ID N:1 or SEQ ID N:2, and peptidyl fragment of the insulin precursor containing chain A and chain B of human insulin. C-end of the first peptidyl fragment is bound with N-end of peptidyl fragment of insulin precursor using residue Arg or Lys. Insulin is prepared by contacting the expressed chimeric protein with chaotropic agent or mercaptan. Invention provides preparing the insulin precursor in bioactive conformation and to screen a peptidyl fragment able to improve stacking the insulin precursor.

EFFECT: improved preparing and identifying method.

16 cl, 3 dwg, 1 ex

Description

1.1. FIELD OF THE INVENTION

This invention relates to a chimeric protein having a sequence similar to that of an intramolecular “mentor” (IMC) linked to a targeted protein. In particular, this invention relates to a chimeric protein containing an IMC-like sequence linked to an insulin precursor. The present invention also relates to a method for producing a correctly laid chimeric protein containing an insulin precursor comprising, inter alia, contacting an improperly laid chimeric protein having an IMC-like sequence linked to an insulin precursor with at least one chaotropic auxiliary agent (additive). In addition, this invention relates to an analysis to screen the amino acid sequence for the ability to improve folding of the insulin precursor using a chimeric protein containing an IMC-like sequence linked to the insulin precursor.

1.2. State of the art

1.2.1. Intramolecular "mentors" and protein folding

Molecular “mentors” are defined as a class of proteins that facilitate the proper folding of other polypeptides, but are not components of a functional assembled structure (Shinde and Inouye, TIBS, 1993, 18: 442-446). Intramolecular “mentors” (IMCs) are part of the precursors of targeted proteins to be twisted, and in their absence, molecules of targeted proteins do not have sufficient information for proper self-folding (self-twisting) (Inouye and Enzyme 1991, 45: 314-321). Unique features of IMC include: a) IMC and the targeted protein are linked by a peptide bond to form a separate polypeptide; B) IMC is indispensable for the formation of an active conformation of the targeted protein, but is not required for the functioning of the targeted protein; c) upon completion of folding (twisting), the IMC protein is removed either by autoprocessing or by another endopeptidase; d) IMC does not function as a catalyst, i.e. one IMC molecule is capable of promoting only one targeted protein molecule; and e) IMC is a highly specific adapted polypeptide that works only in the case of targeted proteins (Inouye, Enzyme, 1991, 45: 314-321).

It was shown that IMC or propeptide can intermolecularly help the targeted protein curl, i.e. The IMC or polypeptide is not bound to a protein-targeted peptide bond, but rather is introduced into the twisting reaction as a separate peptide (US Pat. No. 5,791,021). However, it should be noted that the propeptide used in US Pat. In addition, the intermolecular reaction described in US patent 5719021, must be carried out in a buffered ionic aqueous medium, more suitable for hydrophobic interaction.

Examples of IMC include propeptides of subtilisin, a-lytic protease, carboxypeptidase Y and ubiquitin (Shinde and Inouye, TIBS, 1993, 18: 442-446). Some features of the subtilisin IMC sequence include: a) IMC contains a higher percentage of charged amino acid residues than the targeted protein; b) the distribution of these charged residues within the IMC is extremely uneven, i.e. the N-terminal half contains more positively charged residues than negatively charged residues, and the C-terminal half contains more negatively charged residues than positively charged residues; c) the residues Ser and Thr in the IMC are always unevenly distributed; d) IMC contains a reactively high proportion of aromatic residues; and e) IMC contains a hydrophobic sequence of 9 residues (Inouye, Enzyme, 1991, 45: 314-321). A similar tendency to charged residues is also observed in the a-lytic protease and carboxypeptidase Y (Inouye, Enzyme, 1991, 45: 314-321).

1.2.2. Amino Acid Sequence of Mature Human Growth Hormone

The amino acid sequence of mature human growth hormone (hGH) is described in: Ikehara et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 5956-5960. There is no evidence in the literature (technology) that mature hGH or any part thereof may function as an IMC or propeptide. Indeed, mature hGH or any part of it cannot be considered a propeptide at all, since, by definition, any pre-, pro- or prepro sequence is removed from the mature sequence.

1.2.3. The structure of human insulin

Insulin is a well-defined protein with a known amino acid sequence and structural characteristics (Watson et al., Recombinant DNA - A Short Course; Scientific American Books, WH Freeman Co., New York, 1983, pp. 231-235; Norman and Litwack, In Hormones, Academic Press, New York, 1987, pp. 264-317). This hormone consists of two separate peptide chains, which are an A chain (21 amino acids) and a B chain (30 amino acids) linked by disulfide bridges, as shown in FIG. 1B. Proinsulin is a biological precursor of insulin and is a separate chain formed when chains A and B are linked using a C-peptide (Fig. 1A).

1.2.4. Recombinant DNA human insulin

Human insulin was the first animal protein obtained in bacteria with a sequence identical to that of the human pancreatic peptide (Watson et al., Recombinant DNA - A Short Course; Scientific American Books, WH Freeman Co., New York, 1983, pp. 231-235 ) The first successful expression of human insulin in the laboratory was announced in 1978, and human insulin was approved as a therapeutic drug in 1982 (Johnson, Science, 1983, 219: 632-637).

1.2.4.1. Double stranded method

According to this method, each insulin chain is obtained as a fusion protein with b-galactosidase (b-gal) separate fermentation using E. Coli transformed with plasmids having the DNA sequence encoding human insulin chain A or B, respectively. Products are intracellular and are visible in prominent cytoplasmic inclusion bodies (Williams et al., Science, 1982, 215 (5): 687-689). Recombinant proteins produced in E. Coli typically correspond to 10-40% of the total protein (Burgess, Protein Engineering; Oxender, DL, Fox, CF, Eds .; Alan R Liss, Inc .; New York, 1987; pp. 71- 82).

After isolation from the Taurus, chemical cleavage with CNBr at the Met residue between b-galactosidase and chain A or B, followed by purification, separate A and B peptides are obtained. The peptides are then combined and twisted the AB chain (S-sulfonate forms) in a 2: 1 ratio in the presence of a limited amount of mercaptan to produce an active hormone (Chance et al., In Peptides: Synthesis-Structure-Function, Rich DM, Gross, E., Eds., Pierce Chemical Co., Rockford, II 1981, pp. 721-728; Frank and Chance, In Quo Vadis? Therapeutic Agents Produced by Genetic Engineering, Joyesuk et al., Eds., Sanoff Group, Toulouse- labege, trance, 1985, pp. 137-148). After 24 hours, the yield is approximately 60% based on the amount of B chain taken (Chance et al., In Insulins, Growth Hormone and Recombinant DNA Technology, Raven Press, New York, 1981, pp. 71-85; Johnson, Fluid Phase Equilib., 1986, 29: 109-123). Goeddel et al., Proc. Natl. Acad. Sci. U.S.A., 1979, 76 (1): 106-110, obtained similar results with 20% of the total cellular protein expressed as a fusion protein of chain A or chain B. Subsequent twisting of S-sulfonate chains gives 50-80% of the correct folding.

The large size of the b-gal fusion protein limits yields, since the b-gal fusion protein ( ^- 1000 amino acids) and the insulin A or B chain (21 amino acids or 30 amino acids, respectively) become isolated from the cell ribosome (premature chain termination during translation) and therefore provides incomplete insulin peptides (Burnett, Experimental Manipulation of Gene Expression, Inouye, Ed., Academic Press, New York, 1983, pp. 259-277; Hall, Invisible Frontiers - The Race to Synthesize a Human Gene, Atlantic Monthly Press, New York, 1987). The main improvement of this method is the use of tryptophan (Trp) operon instead of the lac operon (b-gal system) to produce a smaller fusion protein. Trp-operon contains a series of five bacterial genes that sequentially synthesize the enzymes responsible for tryptophan anabolism. One of these enzymes, Trp E, contains only 190 amino acids, compared to 1000 amino acids for b-gal. The Trp E gene, followed by the A or B genes of insulin chains, has additional advantages for increasing the production of fusion protein from 5-10% to 20-30% of the total protein (Hall, Invisible Frontiers - The Race to Synthesize a Human Gene, Atlantic Monthly Press , New York, 1987), since the Trp promoter is a strong promoter in E. Coli. This ultimately leads to 10 times higher expression of the polypeptide compared to the lac (i.e., the b-gal system (Burnett, Experimental Manipulation of Gene Expression, Inouye, Ed., Academic Press, New York, 1983, pp. 259-277) Trp-operon is activated (activated) when E. Coli fermentation is tryptophan-depleted (Hall, Invisible Frontiers - The Race to Sinthesize a Human Gene, Atlantic Monthly Press, New York, 1987; Etienne-Decent , In Genetic Biochemistry: From Gene to Protein, Ellis Horwood Limited, Chichester, UK, 1988, pp. 125-127). This feature is useful in the fermentation process, as cell mass can be maximized first, and then, when needed, cell mass product system citations of insulin can be included, allowing enzymatic media to be depleted by Trp.

Upon completion of the fermentation, the cells are regenerated and ground. Cell debris is then separated from inclusion bodies and inclusion bodies are dissolved in a solvent, although specific details are not known (Wheelwright, Protein Purification, Oxford University press; New York, 1991, p. 217). Inclusion bodies are sometimes dissolved in 6 M hydrochloric acid guanidine and 0.1 mM dithiothreitole (Burgess, Protein Engineering; Oxender, D.L., Fox, C.F., Eds .; Alan R Liss, Inc .; New York, 1987; pp. 71-82). Next, the Trp-LE-Met-A chain and the Trp-LE-Met-B chain are cleaved with CNBr to isolate insulin chains A and B. Further modifications of A and B chains include oxidative sulfitolysis, purification, and compound (combination) to produce crude insulin. Crude insulin is subjected to ion exchange, molecular size and reverse phase high performance liquid chromatography (RP HPLC, RP HPLC) to obtain purified recombinant human insulin (Frank and Chance, Munch Med. Wschr, 1983, 125 (Suppl. 1): 514-520 )

1.2.4.2. Proinsulin method (intracellular)

Human insulin can also be obtained using recombinant microorganisms that produce intact proinsulin, instead of the separate chains of A and B (Kroeff et al., J. Chromatogr., 1989, 481: 45-61). First, mRNA is replicated to cDNA, and the methionine codon is synthesized and attached to the 5'-end of proinsulin cDNA. cDNA is inserted into the bacterial gene in a plasmid vector, which is introduced and then grown in E. coli. Proinsulin can be isolated from a fragment of a bacterial enzyme (b-gal) (or, alternatively, from Trp-LE / Met proinsulin (Trp proinsulin) during the destruction of the methionine linker. The proinsulin chain is subjected to a twisting process to form the correct intramolecular disulfide bridges, and The C-peptide can then be cleaved with enzymes to produce human insulin (Frank and Chance, Munch Med. Wschr, 1983, 125 (Suppl.1): 514-520). In comparison with this method, the previously described double-stranded method is more complicated.

Dorschung et al. constructed a recombinant plasmid encoding the fusion proteins containing mini-proinsulin (B-Arg-A), expressed the fusion proteins in E. Coli (inclusion body) and yeast (secreted), obtained correctly twisted mini-proinsulin by BrCN cleavage and oxidative sulfitolysis and correctly twisted mini-proinsulin was converted into human insulin by trypsin and carboxypeptidase B treatment (EP 0347781 B1; Israeli patent 9562511 B and Australian patent 611303 B2).

Tottrup and Carlsen, Biotechnol. Bioeng., 1990, 35: 339-348 used a yeast system in optimized batch fed fermentation; it was reported that the yields of superoxide dismutase-human proinsulin fusion protein (SOD-PI) were 1,500 mg / L. SOD-PI is proposed as a starting material for the production of recombinant human insulin; yields of the final product were not reported.

Recently, Castellanos-Serra et al., FEBS Letters, 1996, 378: 171-176 expressed in E. Coli a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide (1-22 amino acid residues) linked to the N-terminus of proinsulin using a lysine residue. Chimeric proinsulin was isolated from inclusion bodies, unwound by oxidative sulfitolysis and then converted into insulin as correctly fused proteins by continuous reaction with trypsin and carboxypeptidase B. The IL2-proinsulin fusion protein can be correctly twisted without first removing the IL2 fragment, thus eliminating the stages of cyanogen bromide and related cleaning. However, the stages of oxidative sulfitolysis and the associated purification cannot be avoided using the IL2-proinsulin fusion protein.

1.2.4.3. Proinsulin method (secreted proinsulin)

Villa-Komaroff et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75 (8): 3727-3731 were the first to describe the secretion system of human proinsulin in E. Coli. Thim et al. built recombinant plasmids encoding fusion proteins containing a modified leader sequence of yeast al-factor mating factor and insulin precursor (Thim et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 6766-6770). The leader sequence serves to direct the fusion protein along the secretory pathway of exchange in yeast cells and to expose the fusion protein to the Lys-Arg enzyme processing system. Partial processing (processing) also occurred in one or both dibasic sequences between chains B and A in insulin and other insulin precursors containing a short spacer peptide (consisting of 6 or more amino acid residues) instead of a C peptide. In contrast, no processing is observed in the absence of a spacer peptide in the insulin precursor molecule, for example B-Arg-Arg-A (where A and B are A and B chain of human insulin, respectively). This type of single-chain insulin precursor can be enzymatically converted to insulin by trypsin and carboxypeptidase B. Diers et al., Drug Biotechnjlogy Regulation (Scientific Basis and Practices), Chiu and Gueriguan, Eds., Marcel Dekker, Inc., New York, 1991, pp. . 167-177, describe the untwisted peptide as a leader sequence or segment, then the Lys-Arg sequence, B chain (amino acids 1-29), a short peptide bridge followed by A chain (amino acids 1-21). In this precursor, insulin amino acid 29 of chain B is linked to amino acid 1 of chain A by a short binding peptide containing one basic amino acid connected to the A chain. Human insulin is obtained by transpeptidylation followed by hydrolysis of the resulting ester bond. For the purpose of additional purification, it is chromatographed several times.

1.2.5. The masonry of insulin precursors

Human insulin is a protein having two amino acid chains containing a total of 51 amino acid residues. Six cysteine residues are present in two amino acid chains, in each case two cysteine residues are linked to each other via a disulfide bond. Statistically, there are 15 possibilities for the formation of disulfide bridges within one molecule of human insulin. However, only one of the 15 possibilities is realized in biologically active human insulin with the following disulfide bridges: 1) A6-A11; 2) A7-B7; and 3) A20-B19.

The formation of disulfide bridges that are present in human insulin is affected by an intermediate, and the cysteine residues of human insulin are provided by a sulfur-containing protecting group, for example S-sulfonate (-S-SO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ ) -group (EP 0037255). In addition, pig proinsulin, in which cysteine residues are present in the form of thiol (SH) -containing residues, was used to produce proinsulin with properly connected cysteine bridges (Biochemistry, 1968, 60: 622-629). Obermeier et al. described a method for producing proinsulin with correctly connected cysteine bridges from the corresponding amino acid chain of proinsulin with a concentration of 0.05 to 0.3 g per liter in the presence of mercaptan, chaotropic excipients and hydrophobic adsorbent polymers (US patent 5473049). The oxidative sulfitolysis step is excluded from the process described in US Pat. No. 5,473,049. However, the insulin protein can curl only at low concentrations, which greatly reduces the industrial significance of this process. In addition, the use of large quantities of mercaptan and hydrophobic absorbent polymers complicates the process and increases the cost of wastewater treatment. From the description in US Pat. No. 5,473,049, it is not clear whether the benefit of eliminating the oxidative sulfitolysis step exceeds the increased cost of wastewater treatment.

The citation of the above links should not be interpreted as the assumption that these links represent the state of the art for the present invention.

2. SUMMARY OF THE INVENTION

This invention relates to a chimeric protein comprising from the N-terminus to the C-terminus: a) a first peptide fragment consisting of an amino acid sequence that is at least 40% identical to a domain containing at least 20 N-terminal amino acids of the human growth hormone protein (hGH), in which the percentage of identity is determined over the amino acid sequence identical in length to the hGH domain; b) an Arg residue, or a Lys residue, or a second peptide fragment consisting of at least 2 amino acids, with the largest C-terminal amino acid residue in this peptide fragment being Arg or Lys; and c) a third peptide fragment consisting of an amino acid sequence containing more than two cysteine (Cys) residues, wherein the peptide fragment is not part of the hGH protein. In particular, the invention relates to a chimeric protein in which the third peptide fragment is a precursor of insulin.

The invention also relates to a method for producing a first correctly twisted chimeric protein containing an insulin precursor comprising contacting an incorrectly twisted second chimeric protein containing an insulin precursor, said second chimeric protein containing an insulin precursor consisting of a similar intramolecular “mentor” ( IMC-like) fragment, separated from the precursor of insulin by one or more cleavable amino acid residues, with at least one ha tropic adjuvant (additive) in an aqueous medium; wherein said IMC-like peptide fragment: a) contains from about 20 to about 200 amino acid residues; b) is not a precursor of insulin or part thereof; and c) improves the twisting of the insulin precursor in such a way that the yield of a correctly twisted first chimeric insulin precursor, when the improperly twisted second chimeric protein containing the insulin precursor is contacted with a chaotropic additive, is higher than the yield of a correctly twisted insulin precursor in in the case where an improperly twisted insulin precursor that does not contain the indicated IMC-like peptide fragment is contacted with the same chaotropic additive.

Further, this invention relates to an analysis to screen the amino acid sequence for the ability to improve folding of the insulin precursor, which consists in: a) changing the amino acid sequence of the first peptide fragment of the chimeric protein described in chapter 4.2, which contains the insulin precursor, obtaining the specified chimeric protein with these changes contacting said chimeric protein with said changes with at least one chaotropic additive in an aqueous medium under y and for a time sufficient to ensure that the specified chimeric protein is twisted correctly, and measuring the yield when the specified chimeric protein is twisted with the indicated changes; b) obtaining a chimeric protein used in step (a), but without any changes in the amino acid sequence described in step (a), contacting the chimeric protein without any changes in the amino acid sequence described in step (a), c the same chaotropic additives used in stage (a) in an aqueous medium under the same conditions and during the same time as in stage (a), and measuring the yield upon twisting of the chimeric protein; and c) comparing the twisting yield of the chimeric proteins measured in step (a) and (b), respectively, while the yield in step (a), which is practically equal to or higher than the yield in step (b), indicates that the amino acid the sequence improves the folding (twisting) of the insulin precursor.

Figa and 1B. Structures of proinsulin and mature insulin with properly formed disulfide bridges. Figure 1A depicts the structure of proinsulin. Figure 1B depicts the structure of mature insulin with properly formed disulfide bridges.

Figure 2. Map of the expression vector of hGH-mini-proinsulin (SEQ ID N0: 6) (pZRhi-1).

4. DETAILED DESCRIPTION OF THE INVENTION

Recombinant DNA methods allow the production of human proinsulin or proinsulin with an amino acid sequence and / or amino acid chain length different from the amino acid sequence and / or amino acid chain length of natural human insulin in microorganisms. One major problem in the preparation of human proinsulin or its derivatives in microorganisms such as E. Coli is intracellular cleavage (Ladisch and Kohlman, Biotechnol. Prog., 1992, 2: 469-478). In addition, human proinsulin or its derivatives obtained by the method of recombinant DNA in microorganisms do not contain properly connected cysteine bridges (US patent 5473049).

The process of obtaining human insulin, known before the creation of this invention, is based on the following operations: 1) fermentation of microorganisms transformed with a vector expressing a fusion protein containing human proinsulin or its derivatives; 2) grinding of cells; 3) isolation of fusion protein; 4) cleavage of the fusion protein with cyanogen bromide (cyanogen bromide); 5) isolation of a cleavage product containing a proinsulin sequence; 6) oxidative sulfitolysis; 7) the formation of properly connected cysteine bridges; 8) desalination of proinsulin; 9) chromatographic purification of proinsulin with properly connected cysteine bridges; 9) the concentration of the proinsulin solution; 10) chromatographic purification of a concentrated solution of proinsulin; 11) enzymatic cleavage of proinsulin to produce human insulin; and 12) chromatographic purification of the resulting human insulin (EP 0055945). The disadvantages of this method include numerous stages and losses at the stages of purification, which leads to a low yield of insulin. At the stages of fusion protein isolation, cyanogen bromide cleavage, sulfitolysis and proinsulin purification, losses of proinsulin up to 40% are expected (EP 0055945). On the other hand, the yield of insulin or its derivatives obtained by the recombinant method can be significantly increased if the number of necessary steps is drastically reduced.

One objective of the present invention was to provide a recombinant method for producing human insulin with properly connected cysteine bridges with fewer stages and, therefore, giving a higher yield of human insulin. Another objective of this invention was to create a chimeric protein containing an insulin precursor that can be used in the above method. Another objective of the present invention was to provide a method for screening an amino acid sequence that, when linked to an insulin precursor via a peptide bond, would improve the folding (folding) of the insulin precursor.

Applicants have studied peptide sequences that not only protect the insulin sequence from intramolecular destruction by the host microorganism, but also, in comparison with the existing human insulin expression system, have the following advantages: being linked to the insulin precursor via a peptide bond, 1) initiate twisting of the fused insulin precursor ; 2) facilitate the solubility of the fusion protein and reduce intramolecular interactions in the fusion proteins, thereby allowing twisting of the fused insulin precursor at a high concentration of industrial importance; 3) exclude the stage of splitting with bromine cyanide, oxidative sulfitolysis and the associated stages of purification; and 4) exclude the use of high concentrations of mercaptan or hydrophobic absorbent polymers.

Applicants have unexpectedly discovered that the binding of an IMC-like sequence to an insulin precursor via one or more cleavable amino acid residues serves the purpose of the present invention. An IMC-like sequence has certain features of an IMC sequence, such as promoting twisting of the targeted protein, a higher percentage of charged amino acid residues than the targeted protein, the presence of a “polarized” distribution of charged amino acid residues, and the presence of a sequence that seems to be adapted specifically for targeted protein. However, the IMC-like sequence used in this invention differs from the IMC sequence in some basic aspects. First, the IMC-like sequence is heterogeneous with the targeted protein, i.e. not with a targeted protein propeptide. For example, if the insulin precursor is a targeted protein that needs to be twisted, the IMC-like sequence is not an insulin precursor or fragment thereof. In addition, the length of the IMC-like sequence is approximately 20 to 200 amino acid residues.

Further, contrary to what is claimed in the prior art (Castellanos-Serra et al., FEBS Letters, 1996, 378: 171-176), applicants have unexpectedly discovered that the inclusion in the IMC-like sequence of one or more cleavable amino acid residues, identical to one or more cleavable amino acid residues that separate the IMC-like sequence and the insulin precursor, after twisting, remove the IMC-like sequence in fragments, thereby simplifying the down-stream treatment steps.

For simplicity of description, the description of the invention is divided into the following subsections.

4.1. Nucleic acids encoding a chimeric protein described in Section 4.2.

The present invention encompasses an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein described in Section 4.2.

In a particular embodiment, the invention provides an isolated nucleic acid having a nucleotide sequence encoding a chimeric protein having the sequence of SEQ ID NO: 6.

In another particular embodiment, the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein having the sequence of SEQ ID NO: 7.

In a preferred embodiment, the invention encompasses isolated DNA with a nucleotide sequence encoding a chimeric protein described in Section 4.2.

In another preferred embodiment, the present invention encompasses an isolated nucleic acid with a nucleotide sequence complementary to the nucleotide sequence encoding a chimeric protein described in Section 4.2.

In another particular embodiment, the invention encompasses an isolated nucleic acid capable of hybridizing to a nucleotide sequence encoding the first, second, and third peptide fragments of the encoded DNA of a chimeric protein described in Section 4.2.

A nucleic acid containing a nucleotide sequence encoding a chimeric protein described in Section 4.2., Or any of its fragments, analogues or derivatives, can be obtained by any method (s) known in the art (s). Nucleic acid can be completely synthesized chemically. Or nucleic acid encoding each chimeric protein fragment, i.e. the first, second or third peptide fragment can be obtained by molecular cloning or can be purified from the desired cells. The nucleic acid encoding each chimeric protein fragment can then be ligated chemically or enzymatically to form a nucleic acid with the nucleotide sequence encoding the chimeric protein described in Section 4.2., Or any fragments, analogs or derivatives thereof.

Any human cell could potentially serve as a source of nucleic acid to isolate a nucleic acid encoding hGH. Any mammalian cell can potentially serve as a source of nucleic acid for the isolation of nucleic acids encoding insulin. The nucleotide sequences encoding insulin can be isolated from a mammal, human, pig, cow, feline, bird, horse, canine, as well as from rodents or primates, etc.

DNA can be obtained by standard methods known in the art from cloned DNA (e.g., a DNA-based library), chemical synthesis, cloning of cDNA, or cloning of genomic DNA or fragments thereof purified from desired cells (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, DM (ed), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, UK Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron regions of DNA, in addition to coding regions; cDNA clones will contain only exons. Regardless of the source of the genes, they are molecularly cloned into the corresponding vector for the purpose of (propagating) the cultivation of the gene.

In molecular cloning of a gene from cDNA, cDNAs are obtained from totally (completely) cellular RNA or mRNA by methods well known in the art. Genes can also be obtained from genomic DNA, where DNA fragments are obtained (for example, using restriction enzymes or hydrodynamic fragmentation), some of which encode a given gene. Linear DNA fragments can then be size-separated by standard methods, including, without limitation, agarose and polyacrylamide gel electrophoresis and column chromatography.

When a nucleic acid containing the nucleotide sequence encoding the chimeric protein described in Section 4.2., Or any fragments, analogs or derivatives thereof, is obtained, its identity can be confirmed by nucleic acid sequencing (by any method well known in the art) and by comparison with known sequences. DNA sequence analysis can be carried out by any method known in the art, including, without limitation, the Maxam and Gilbert method (Maxam and Gilbert, 1980, Meth. Enzymol. 65: 499-560), the Sanger dideoxy method (Sanger et al. , 1977, Proc. Natl. Acad. Sci. USA, 74: 5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent 4,795,699), the use of an automatic DNA sequencer (e.g., Applied Biosystems, Foster City, CA) or the method described in International application WO 97/15690.

Nucleic acids hybridizing to a nucleic acid containing a nucleotide sequence encoding a chimeric protein described in Section 4.2., Or any fragments, analogs or derivatives thereof, can be isolated by hybridization with a nucleic acid under relaxed conditions, under stringent conditions, or under conditions of moderate stringency (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA, 78: 6789-6792).

4.2. Chimeric protein

The present invention encompasses a chimeric protein containing, from the N-terminus to the C-terminus: a) a first peptidyl fragment consisting of an amino acid sequence that is at least 40% identical to a domain containing at least 20 N-terminal amino acids of the human growth hormone protein (hGH), in which the percentage of identity is determined over the amino acid sequence identical in length to the hGH domain; b) an Arg residue, or a Lys residue, or a second peptidyl fragment consisting of at least 2 amino acids, wherein in this peptidyl fragment the largest C-terminal amino acid residue is Arg or Lys; and c) a third peptidyl fragment consisting of an amino acid sequence containing more than two cysteine (Cys) residues, wherein this peptidyl fragment is not part of the hGH protein.

In a preferred embodiment, the invention includes a chimeric protein as described above, wherein the first peptidyl fragment consists of an amino acid sequence at least 60% identical to the domain of the hGH protein.

In another preferred embodiment, the invention includes a chimeric protein as described above, wherein the first peptidyl moiety is capable of binding to an anti-hGH antibody.

In a more preferred embodiment, the present invention provides a chimeric protein as described above, wherein the first peptidyl fragment consists of the amino acid sequence of SEQ ID NO: l.

In another, more preferred embodiment, the present invention provides a chimeric protein as described above, wherein the first peptidyl fragment consists of the amino acid sequence of SEQ ID NO: 2.

In a preferred embodiment, the invention includes a chimeric protein as described above, wherein the second peptidyl moiety consists of the amino acid sequence of SEQ ID NO: 3.

In a particular embodiment, the invention includes a chimeric protein as described above, wherein the third peptidyl moiety is a precursor to insulin, preferably of human origin.

In a more preferred embodiment, the invention includes a chimeric protein as described above, wherein the human insulin precursor is capable of binding to an anti-human insulin antibody.

In another, more preferred embodiment, the invention encompasses the chimeric protein described above, wherein the human insulin precursor has the amino acid sequence of SEQ ID NO: 4.

In yet a more preferred embodiment, the present invention encompasses the chimeric protein described above, wherein in the human insulin precursor, chain B and chain A of the human insulin precursor are separated by an amino acid residue or a peptidyl fragment containing from 2 to 34 amino acid residues.

In another, more preferred embodiment, the present invention encompasses the chimeric protein described above, wherein the human insulin precursor has the amino acid sequence of SEQ ID NO: 5.

In a most preferred embodiment, the invention includes a chimeric protein containing the amino acid sequence of SEQ ID NO: 6.

In another most preferred embodiment, the invention includes a chimeric protein containing the amino acid sequence of SEQ ID NO: 7.

4.3. Obtaining a chimeric protein described in Section 4.2.

Chimeric proteins described in Section 4.2. And their derivatives, analogues and fragments can be obtained by any method known in the art, including, without limitation, recombinant expression methods, isolation from natural sources, and chemical synthesis.

For example, chimeric proteins described in Section 4.2. Can be obtained by recombinant expression of a recombinant protein. For recombinant expression, the gene or region of the gene encoding the chimeric proteins described in Section 4.2. Is inserted into the corresponding cloning vector for expression in a particular host cell. You can use a large number of vector host systems known from the prior art. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages, such as lambda derivatives, or plasmids, such as pBR322 or pUC plasmid derivatives or a Bluescript vector (Stratagene). Insertion into a cloning vector can, for example, be accomplished by subsidizing a DNA fragment into a cloning vector that has complementary “sticky” ends. However, if the complementary restriction sites used for DNA fragmentation are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Or any given site can be obtained by ligating nucleotide sequences (linkers) with DNA ends; these subsidized linkers may be specific chemically synthesized oligonucleotides encoding restriction enzyme recognition sites (sequences). Recombinant molecules can be introduced into host cells by transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are formed.

In an alternative method, a given gene can be identified and isolated after insertion into the corresponding cloning vector using the "shotgun" method. Prior to insertion into a cloning vector, enrichment with a given gene can be carried out, for example, size fractionation.

In specific embodiments of the invention, the transformation of the host cells of the recombinant DNA, which introduces as an insert the selected gene encoding the chimeric proteins described in Section 4.2., By a cDNA or synthesized DNA sequence makes it possible to obtain multiple copies of this gene. Those. the gene can be obtained in large quantities by growing transformants, isolating recombinant DNA from transformants and, if necessary, returning the inserted gene from the isolated recombinant DNA.

The nucleotide sequence encoding the chimeric proteins described in Section 4.2., And their derivatives, analogs and fragments, or their functionally active analogues, or fragments, or other derivatives, can be inserted into a suitable expression vector, i.e. into a vector that contains the necessary elements for transcription and translation of the inserted protein-coding sequence. A number of host vector systems can be used to express a protein coding sequence. These systems include, without limitation, mammalian cell systems infected with a virus (eg, vaccinia virus, adenovirus, etc.); virus insect cell systems (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Expression elements of vectors vary in strength and specificity. Depending on the host-vector system used, any of the appropriate transcription and translation elements may be used.

Any of the previously described methods for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene, consisting of the corresponding sequences that control the transcription / translation of signal and coding proteins. These methods may include in vitro recombinant DNA and synthetic methods and in vivo recombinants (genetic recombination). The expression of the nucleotide sequence encoding the chimeric proteins described in Section 4.2., And their derivatives, analogs and fragments, can be regulated by the second nucleotide sequence, so that the chimeric proteins described in Section 4.2. Are expressed in a host transformed with recombinant DNA. For example, the expression of chimeric proteins described in Section 4.2. Can be controlled by any promoter / enhancer known in the art. Promoters that can be used to control the expression of chimeric proteins described in Section 4.2. Include, without limitation, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature, 290: 304-310), promoters contained in a 3 'long Routh sarcoma virus terminal repeat (Yamamoto, et al., 1980, Cell 22: 787-797), herpestimidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 78: 1441-1445), gene regulatory sequences metallothionein (Brinster et al., 1982, Nature, 296: 39-42), prokaryotic expression vectors such as the b-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc, Natl. Acad. Sci. USA, 75 : 3727-3731), the composite promoter (tac) (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. USA, 80: 21-25), or the Trp E promoter (Hall, Invisible Frontiers-The Race to Synthesize a Human Gene, Atlantic Monthly Press, New York, 1978) see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 79-94; yeast or other fungal-based promoters, such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the following transcriptional control regions in animals that exhibit tissue specificity and have been used in transgenic animals: regulatory region an elastase I gene that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987 , Hepatology 7: 425-515), regulatory region of the insulin gene active in pancreatic Sgiach beta cells (Hanahan, 1985, Nature 315: 115-122). an immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., Mol. Cell. Biol., 7: 1436-1444), the regulatory region of the vector based on the breast tumor virus, lymphocytes and mast cells (Leder et al., 1986, Cell 45: 485-495), the regulatory region of the albumin gene, active in the liver (Pinkert et al. , 1987, Genes and Devel. 1: 268-276), a regulatory region of the alpha-fetoprotein gene active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58). the regulatory region of the alpha 1-antitrypsin gene active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), the regulatory region of the beta globin gene active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94), the regulatory region of the gene of the main myelin protein active in brain oligodendrocytes (Readhead et al., 1987, Cell 48: 703-712), the regulatory region the myosin light chain-2 gene active in skeletal muscle (Sani, 1985, Nature 314: 283-286) and the regulatory region of the gonadotropin-releasing hormone gene active in the hypothalamus (Mason et al., Science 234: 1372-1378).

For example, you can use a vector that contains a promoter operably linked to a nucleic acid encoding the chimeric proteins described in Section 4.2., One or more origin of replication and, if necessary, one or more selective marker (e.g., antibiotic resistance gene). Expression vectors containing generations of genes encoding chimeric proteins described in Section 4.2., Or fragments, analogs or derivatives thereof, can be identified by three general methods: (a) nucleotide hybridization, (b) the presence or absence of the function of the marker gene, and ( c) expression of the inserted sequences. In the first method, the presence of the gene encoding the chimeric proteins described in Section 4.2., Embedded in the expression vector, can be determined by hybridization of nucleic acids using probes containing sequences homologous to the inserted gene encoding the chimeric proteins described in Section 4.2. In the second method, the recombinant vector / host system can be identified and selection can be made based on the presence or absence of certain functions of the marker gene (for example, thymidine kinase activity, antibiotic resistance, phenotype resulting from transformation, formation of inclusion bodies in baculovirus, etc. .), due to the insertion of the gene encoding the chimeric proteins described in Section 4.2., in the vector. For example, if a gene encoding the chimeric proteins described in Section 4.2. Is inserted into the sequence of the marker gene of the vector, recombinants containing an insert encoding the chimeric proteins described in Section 4.2. Can be identified by the absence of the function of the marker gene. In a third method, recombinant expression vectors can be identified by analyzing the products of chimeric proteins expressed by the recombinant. Such assays may be based, for example, on the physical and functional properties of chimeric proteins described in Section 4.2., In in vitro test systems, for example, on binding to an anti-hGH antibody or against insulin.

When a specific recombinant DNA is identified and isolated, several techniques are known in the art that can be used to culture it. When a suitable host system and growth conditions are established, recombinant expression vectors can be cultured and quantified. As explained above, expression vectors that can be used include, without limitation, the following vectors or their derivatives; vectors based on animal and human viruses such as vaccinia or adenovirus; vectors based on insect viruses such as baculovirus; yeast vectors, bacteriophage vectors (e.g. lambda) and plasmid and cosmid DNA vectors (few are listed).

In addition, you can choose a strain of host cells that modulates the expression of the inserted sequences or modifies and processes the gene product in a specific, predetermined manner. Expression using certain promoters may be increased in the presence of certain inducers; thus, the expression of the recombinant DNA-derived chimeric protein described in Section 4.2. can be controlled. Moreover, various host cells have characteristic and specific mechanisms of translational and post-translational processing and modification (for example, glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems can be selected to guarantee the desired modification and processing of a foreign expression protein. For example, expression in a bacterial system can be used to produce a non-glycosylated nuclear protein product. Expression in yeast gives a glycosylated product. Expression in mammalian cells can be used to guarantee "native" glycosylation of a heterologous protein. In addition, different vector-host expression systems can affect processing reactions to varying degrees.

It is possible to carry out the cloning and expression of both DNA and genomic sequences.

The chimeric protein described in Section 4.2., Or fragments, analogs or derivatives thereof, can also be isolated and purified by standard methods, including chromatography (e.g., column, ion exchange, affinity, and molecular size chromatography), centrifugation, various solubilities, or other standard purification methods proteins. Functional properties can be determined using any suitable analysis method.

The nucleotide sequence encoding a chimeric protein described in Section 4.2, or fragments, analogs or derivatives thereof, can be mutated in vitro or in vivo to create and / or destruction of translation, initiation or termination sequences (sites) or for changes in coding regions . Any mutagenesis methods known in the art may be used, including, without limitation, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551). the use of TAB linkers (Pharmacia) containing mutations of PCR primers, etc.

The experimental work associated with mutagenesis consists, firstly, of site-directed mutagenesis followed by phenotypic testing of the altered gene product. Some of the most commonly used site-directed mutagenesis protocols use predominantly vectors that can produce single-stranded as well as double-stranded DNAs, if necessary. Typically, the mutagenesis protocol with such vectors is as follows. A mutagenic primer is synthesized, i.e. a primer complementary to a variable sequence, but consisting of one or a small number of modified, introduced or delegated bases. The primer is extended in vitro using DNA polymerase, and after several additional manipulations, double-stranded DNA is now transfected into bacterial cells. Next, the desired mutant DNA is identified by various methods and the target protein is purified from the clone containing the mutant sequence. For longer sequences, additional cloning steps are often required, because in these vectors extended insertions (more than 2 thousand bp) are unstable. Protocols are known to those skilled in the art, and kits for site-directed mutagenesis in large quantities are available from biotechnology companies such as Amersham Life Science, Inc. (Arlington Heights, IL) and Stratagene Cloning Systems (LaJolla, CA).

In addition, the chimeric protein described in Section 4.2., Or fragments, analogs or derivatives thereof, can be chemically synthesized (see, for example, Clark-Lewis et al., 1991, Biochem. 30: 3128-3135 and Merrifield, 1963, J. Amer. Chem. Soc. 85: 2149-2156). For example, chimeric proteins described in Section 4.2., Or fragments, analogs or derivatives thereof, can be synthesized by the solid phase method, cleaved from polymers and purified by high performance liquid preparative chromatography (e.g. see Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., NY, pp. 50-60). The chimeric protein described in Section 4.2., Or fragments, analogs or derivatives thereof, can also be synthesized using a peptide synthesizer. The structure of synthetic peptides can be confirmed by amino acid analysis or sequencing (e.g. Edman cleavage; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N. Y., pp. 34-49).

Chimeric proteins described in Section 4-2., Or fragments, analogs or derivatives thereof, can be synthesized completely by sequential addition of amino acid residues or as subcomponents of fragments that can be composed using methods well known in the art, such as for example, condensation of fragments (Shin et al., 1992, Biosci. Biotech. Biochem. 56: 404-408; Nyfeler et al., 1992, Peptides, Proc. 12th Amer. Per. Soc., Smith and Rivier (eda), Leiden, pp. 661-663; and Nokihara et al., 1990, Protein Research Foundation, Yanaihara (ed), Osaka, pp. 315-320).

In a less preferred embodiment of the invention, the chimeric proteins described in Section 4.2., Or fragments, analogs or derivatives thereof, can be obtained by proteolysis of the protein, followed by purification using standard methods, such as those described above (e.g., immunoaffinity purification).

4.4. Obtaining a Correctly Twisted Insulin Precursor

This invention encompasses a method for producing a correctly twisted first chimeric protein containing an insulin precursor by contacting an incorrectly twisted second chimeric protein containing an insulin precursor, said second chimeric protein containing an insulin precursor consisting of an intramolecular “mentor” (IMC-like) a peptide fragment separated from the precursor of insulin by one or more cleavable amino acid residues with at least one chaotropic additive in the aquatic environment; wherein said IMC-like peptide fragment: a) contains from about 20 to about 200 amino acid residues; b) is not a precursor of insulin or a fragment thereof; and c) improves the folding of the insulin precursor, such that the yield of a correctly twisted first chimeric protein containing the insulin precursor in the case when the incorrectly twisted second insulin precursor containing the chimeric protein is in contact with a chaotropic additive is higher than the yield of the correctly twisted insulin precursor in in the case when an improperly twisted insulin precursor, not containing an IMC-like fragment, comes into contact with the same chaotropic additive.

As used herein, the term “human insulin precursor” refers to a molecule that 1) contains the A chain and B chain of human insulin or their analogs, derivatives and fragments, 2) contains six cysteine residues, 3) contains a leaving binding fragment connected to A chain and B chain of insulin, and 4) is able to bind to an antibody against human insulin. Examples of the human insulin precursor include, without limitation, the insulin precursors described in Ladisch and Kohlmann, Biotechnol. Prog., 1992, 8: 469-478; Thim et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 6766-6770; U.S. Patent 5,473,049; 5457066; and EP 0347781 B1.

The term “correctly twisted” human insulin precursor or a chimeric protein containing human insulin precursor refers to a molecule that has the conformation and disulfide bridges found in natural biologically active human insulin in the human insulin precursor, i.e. disulfide bridges are formed between a) A-6 and A-11, b) A-7 and B-7, c) A-20 and B-19. The term “improperly twisted” human insulin precursor or a chimeric protein containing an insulin precursor refers to a molecule that lacks conformation, disulfide bridges found in naturally occurring biologically active human insulin, or both.

In a preferred embodiment, the present invention encompasses a method for producing a correctly twisted insulin precursor-containing chimeric protein as described above, the insulin precursor being of “human origin”. It is also preferred that the human insulin precursor can bind to an anti-human insulin antibody. It is still preferred that the human insulin precursor comprises the amino acid sequence of SEQ ID NO: 4. Also preferred in the human insulin precursor is when the B chain and A chain of the human insulin precursor are separated by an amino acid residue or a peptidyl fragment containing from 2 to 34 amino acid residues. More preferably, the precursor of human insulin has the amino acid sequence of SEQ ID NO: 5.

In a preferred embodiment, the invention includes a method for producing a properly twisted insulin precursor-containing chimeric protein as described above, wherein the IMC-like peptidyl moiety has a higher percentage of charged amino acid residues than the insulin precursor. It is also preferred that in the IMC-like peptidyl fragment, the N-terminal half contains more positively charged amino acid residues than negatively charged amino acid residues, and the C-terminal half contains more negatively charged amino acid residues than positively charged amino acid residues. It is further preferred that the IMC-like peptidyl moiety consists of an amino acid sequence that is at least 40% identical to a domain containing at least the first 20 N-terminal amino acids of the human growth hormone protein (hGH), the percent identity being it is determined throughout the amino acid sequence identical in length to the hGH domain. It is also preferred that the IMC-like peptidyl moiety consists of an amino acid sequence of at least 60% identical to the domain containing at least the first 20 N-terminal amino acids of the human growth hormone protein (hGH). Furthermore, it is preferable when the IMC-like fragment is capable of being bound by an anti-hGH antibody. More preferably, when the IMC-like peptidyl fragment has the sequence of SEQ ID NO: l. It is also more preferred that the IMC-like peptidyl fragment consists of the amino acid sequence of SEQ ID NO: 2.

In a preferred embodiment, the present invention includes a method for producing a correctly twisted insulin precursor-containing chimeric protein as described above, wherein the cleavable amino acid residue is an Arg or Lys residue. It is also preferred that the cleavable amino acid residues consist of the amino acid sequence of SEQ ID NO: 3.

In a particular embodiment, the invention includes a method for producing a correctly twisted insulin precursor-containing chimeric protein as described above, wherein in a wrongly twisted insulin precursor-containing chimeric protein, a peptidyl moiety is located at the N-terminus of said chimeric protein. In another such particular embodiment of the invention, an IMC-like peptidyl moiety is located at the C-terminus of said chimeric protein. In yet another particular embodiment of the invention, an IMC-like peptidyl moiety is located between the B chain and the A chain of the insulin precursor.

In a preferred embodiment, the present invention encompasses a method for producing a properly twisted insulin precursor-containing chimeric protein as described above, wherein the IMC-like peptidyl fragment contains one or more cleavable amino acid residues identical to one or more cleavable amino acid residues that separates the IMC-like peptidyl fragment and the insulin precursor in the second chimeric protein containing the insulin precursor. More preferably, the cleavable amino acid residue is an Arg or Lys residue.

In a most preferred embodiment, the present invention includes a method for producing a correctly twisted insulin precursor-containing chimeric protein as described above, wherein the incorrectly twisted insulin precursor-containing chimeric protein consists of the amino acid sequence of SEQ ID NO: 6. It is also most preferred when the improperly twisted second insulin precursor containing chimeric protein consists of the amino acid sequence of SEQ ID NO: 7.

Chaotropic additives are compounds that break the hydrogen bonds in aqueous solutions. In a specific embodiment, the invention encompasses a method for producing a correctly twisted insulin precursor chimeric protein as described above, wherein the chaotropic additive is selected from the group consisting of guanidine hydrochloride, ethylene carbonate, thiocyanate, dimethyl sulfoxide and urea. Urea is a preferred chaotropic agent. More preferably, urea is present in concentrations of about 2.0-8.0 M. Most preferably, urea is present in concentrations of about 3.0-6.0 M.

In a particular embodiment, the present invention includes a method for producing a correctly twisted insulin precursor chimeric protein as described above, in which an improperly twisted second insulin precursor chimeric protein is contacted with at least one chaotropic additive in an aqueous medium at a pH of about 8, 0 to 10.5 and at a concentration of an improperly twisted second chimeric protein containing the insulin precursor, from about 0.05 g to 15.0 g per liter. Preferably, when the pH is maintained in the range of about 9.0 to 10.0. It is also preferred that an improperly twisted second chimeric protein containing the insulin precursor is present at a concentration of about 0.5 to 5.0 g per liter. More preferably, the concentration of the improperly twisted second chimeric protein containing the insulin precursor is from about 2.0 to 3.0 g per liter.

Mercaptans are water soluble compounds containing at least one —SH group. In another specific embodiment, the invention includes a method for producing a correctly twisted insulin precursor containing the above-described chimeric protein, further comprising contacting an incorrectly twisted second insulin precursor containing a chimeric protein with such an amount of mercaptan that produces less than 5-SH mercaptan radicals per cysteine residue an improperly twisted second insulin precursor chimeric protein. In yet another specific embodiment of the present invention, an improperly twisted second insulin precursor containing chimeric protein is in contact with mercaptan and a chaotropic additive. In yet another specific embodiment of the present invention, an improperly twisted second insulin precursor containing chimeric protein is contacted sequentially with mercaptan and a chaotropic additive. Preferably, the amount of mercaptan is such that it provides from about 0.07 to about 1.0 -SH of the mercaptan radical per cysteine residue of an improperly twisted second insulin precursor chimeric protein. It is also preferred that the mercaptan is selected from the group consisting of dithiothreitol, dithioerythrol, 2-mercaptoethanol, cysteine, methylthioglycolate, 3-mercapto-1,2-propanediol and 3-mercaptopropionic acid. More preferably, the mercaptan is 2-mercaptoethanol.

In yet another specific embodiment, the present invention encompasses a method for producing a correctly twisted insulin precursor containing a chimeric protein of the above-described chimeric protein, further comprising separating a correctly twisted first insulin precursor containing a chimeric protein from an incorrectly twisted second insulin precursor containing a chimeric protein. Preferably, the first insulin precursor-containing chimeric protein is separated from the second insulin precursor-containing chimeric protein by ultrafiltration. More preferably, when the ultrafiltration is carried out at a pH of from about 8.0 to 11.0. Most preferably, the ultrafiltration is carried out at a pH of about 9.0 to 10.0.

In yet another specific embodiment, the present invention encompasses a correctly twisted insulin precursor containing a chimeric protein obtained by the above method.

4.5. Screening for an amino acid sequence that improves folding of an insulin precursor

The present invention encompasses a test to screen the amino acid sequence for the ability to improve folding of an insulin precursor, comprising: (a) changing the amino acid sequence of the first peptidyl fragment of a chimeric protein described in Section 4.2., Obtaining said chimeric protein with said changes, contacting said chimeric a protein with the indicated changes with at least one chaotropic additive in an aqueous medium under such conditions and for such a time that bound chimeric protein is twisted right and a determination output at packing (twisting) of said chimeric protein with said changes; (b) obtaining the same chimeric protein that is used in step (a), but without any changes in the amino acid sequence described in step (a), contacting the chimeric protein that does not contain any changes in the amino acid sequence described in step (a) bringing into contact a chimeric protein that does not contain any changes in the amino acid sequence described in step (a) with the same chaotropic additive (s) used on stage (a) in an aqueous medium under the same conditions y and during the same time as in stage (a), and determining the yield when laying the chimeric protein; and (c) comparing the yield during the stacking of chimeric proteins in steps (a) and (b), respectively, wherein the yield in step (a) is substantially equal to or higher than the yield in step (b) indicates that the amino acid sequence improves styling of the insulin precursor.

The amino acid sequence of the first peptidyl moiety described in Section 4.2. Can be altered by any method known in the art for mutagenesis, preferably the mutagenesis methods described in Section 4.3.

In a preferred embodiment of the above test, the chimeric protein consists of the amino acid sequence of SEQ ID NO: 7.

In another preferred embodiment of the above test, the chimeric protein consists of the amino acid sequence of SEQ ID NO: 7.

In a further preferred embodiment of the aforementioned test, the chaotropic additive is urea.

In yet another preferred embodiment of the above test, the test further comprises contacting the chimeric protein, in steps (a) and (b), respectively, with such an amount of mercaptan that produces less than 5 -SH radicals of mercaptan per cysteine residue of the chimeric protein. More preferably, the mercaptan is 2-mercaptoethanol.

In a particular embodiment, the invention encompasses the product of the above test.

5. EXAMPLE

Chemically synthesize a DNA fragment encoding hGH-mini-proinsulin, consisting of the amino acid sequence of SEQ ID NO: 6. The DNA fragment is cloned into a bacterial expression vector under the control of the Trp promoter. An expression vector containing hGH-mini-proinsulin is transformed into E. Coli strain PR1, and recombinant cells are cultured in M9-CA media in the presence of trace elements. HGH-mini-proinsulin fusion proteins are isolated from inclusion bodies and twisting is carried out under such conditions that disulfide bridges form in twisted hGH-mini-proinsulin fusion proteins, as they would form in properly twisted human proinsulin, i.e. disulfide bridges A6-A11, A7-B7 and A20-B19. Twisted hGH-mini-proinsulin fusion proteins are separated from non-twisted proteins by ultrafiltration at 100 kD. Isolated, correctly twisted fused proteins of hGH-mini-proinsulin are subjected to trypsin hydrolysis to form properly twisted Arg (B31) -human insulin. Arg (B31) -human insulin is purified by cation exchange chromatography to approximately 90% purity. The purified Arg (B31) -human insulin is then hydrolytically cleaved with carboxypeptidase B to form properly twisted human insulin, which is then purified by reverse phase HPLC. Thus obtained insulin is characterized by N-terminal sequencing, molecular weight determination and peptide mapping.

5.1. The construction of the expression vector of hGH-mini-proinsulin

A DNA fragment encoding hGH-mini-proinsulin, consisting of the amino acid sequence of SEQ ID NO: 6, is synthesized chemically by the method described by Gan et al., Gene, 1989, 79: 159-166. A 5 ′ Cla I site and a 3 ′ Hind III site are introduced into the synthesized DNA fragment. In short, a fragment of 5 ′ Cla I to 3 ′ Krn I that cuts the nucleotide sequence encoding the amino acid residues 51 and 52 of SEQ ID NO: 6, and a fragment from 5 ′ Cln I to 3 ′ Hind III are chemically synthesized and subcloned into pUCIS vector, respectively. Then the DNA fragment encoding the complete amino acid sequence of SEQ ID NO: 6 is transferred to the modified pATH2 vector so that expression of hGH-mini-proinsulin is controlled by the Trp promoter and the SD sequence. The resulting vector, pZRhi-1 (FIG. 2) is used to express the hGH-mini-proinsulin fusion protein.

5.2. HGH-mini-proinsulin fusion protein expression

The expression vectors of the hGH-mini-proinsulin fusion protein are transformed into E. Coli strain RR1 K12 W3110. Transformed E. Coli cells are cultured in M9-CA media in the presence of trace elements. The expression level of hGH-mini-proinsulin fusion proteins is determined both in the rocking flask and in the fermenter. In both cases, a high level of expression is observed, reaching 20-25% of the total E. Coli proteins.

5.3. Reassembly of the hGH-mini-proinsulin fusion protein

HGH-mini-proinsulin fusion proteins are expressed in an insoluble form called an “inclusion body”. To release inclusion bodies, E. Coli cells are ground in a high pressure homogenizer at 800 bar (80,000 kPa). Cell debris and soluble proteins of E. Coli cells are removed by centrifugation at 10,000 rpm. The sediment of inclusion Taurus containing hGH-mini-proinsulin fusion proteins is washed 3 times with water. The resulting pellets of inclusion bodies, in which the fused proteins of hGH-mini-proinsulin are approximately 90% pure, are used as starting material for twisting. Inclusion bodies are dissolved in 8 M urea, pH 10.4 at a concentration of hGH-mini-proinsulin fusion protein of 20-30 mg / ml in the presence of 2 to 6 mM mercaptoethanol. Insoluble material is removed by centrifugation. The supernatant is diluted 10 times with a solution with a low concentration of urea so as to achieve a final concentration of urea from 3 to 6 M, pH 9-10 and mercaptoethanol from 0.2 to 0.6 mm. A standard stacking operation is carried out at 4 ° C with a urea concentration of 3.2 M, pH 9.3. The laying process (twisting) is monitored by C4 reverse phase HPLC in a gradient of 30-47% acetonitrile in 0.1% phosphate buffer. In this chromatographic mode, the retention time of correctly twisted hGH-mini-proinsulin is about 23 minutes. The stacking (twisting) operation can be completed in 24 hours. The output when repacking is about 70%.

After laying, the mixture is fractionated by ultrafiltration at 100 kD. The correctly twisted hGH-mini-proinsulin fusion proteins found in the filtrate fractions are concentrated using a 10 kD ultrafiltration system. Urea is removed with water at pH 3.5. The output at the stages of ultrafiltration is above 85%.

5.4 Tryptic hydrolysis (trypsinolysis) of correctly twisted hGH-mini-proinsulin after re-laying

When performing tryptic hydrolysis, the concentration of the correctly twisted hGH-mini-proinsulin fusion protein is about 10-12 mg / ml, preferably 10 mg / ml. The ratio of trypsin to hGH-mini-proinsulin fusion protein is in the range of about 1:60 to 1: 250, preferably 1: 100. The pH value is maintained in the range of about 10 to 11, preferably 10.8. The hydrolysis is carried out at 4 ° C. for about 1 to 5 hours, preferably within 3.5 hours. Cleavage (hydrolysis) is stopped by adjusting the pH to 3.5 with a phosphate buffer. Analysis by reverse phase HPLC shows that the yield at this stage of cleavage is above 95%. At pH above 10, trypsin acts on the following Arg residues: the Arg residue between the B and A chains of human mini-proinsulin, the Arg residue between the hGH fragment and the mini-proinsulin fragment, and Arg residues in the hGH fragment. Trypsin hydrolysis gives several small parts of the hGH fragment and human insulin with additional (extra) Arg at the C-terminus of the B chain, which is called Arg (B31) -human insulin. The residue Arg (B22) of human insulin does not cleave under the above conditions, apparently due to steric hindrances caused by (this) three-dimensional structure.

Arg (B31) -human insulin is purified by cation exchange chromatography with NaCl in the presence of 10 mM nitrate buffer as solvent. Purified Arg (B31) -human insulin has a purity of more than 90%.

5.5. Conversion of Arg (B31) -human insulin to human insulin

The residue of Arg (B31) at the C-terminus of Arg (B31) - human insulin is removed using carboxypeptidase B. The concentration of correctly twisted Arg (B31) -human insulin is about 10 mg / ml. The ratio of carboxypeptidase B and Arg (B31) - human insulin is maintained at about 1: 1000. Cleavage (hydrolysis) takes place at 37 ° C for approximately 1 hour in 50 mM Tris-HCl buffer pH 8.0. The hydrolysis is stopped by adjusting the pH to 3.5 with a phosphate buffer. The output of human insulin is more than 99%.

5.6. Human insulin purification

Human insulin obtained by hydrolytic decomposition with carboxypeptidase B was applied to a C8 column for reverse phase HPLC, which was equilibrated with 0.1% phosphate buffer, pH 3.0. Human insulin is eluted with acetonitrile in a gradient of 17-35% in 0.1% phosphate buffer, pH 3.0. The insulin fractions were collected and acetic acid was added until a concentration of 0.125 to 0.2 M, pH 6.0 was reached. The insulin obtained in this way crystallizes at 4 ° C. The purity of human insulin exceeds 99%.

5.7. Characterization of purified human insulin

The first 15 N-terminal amino acids of purified human insulin and WHO standard human insulin determine standard Edman cleavage. The N-terminal sequences of both A and B chains of purified human insulin are identical to the corresponding WHO sequences of standard human insulin.

The molecular weight of purified human insulin and the molecular weight of WHO standard human insulin are determined by mass spectrometry on a VG Platform. The M / Z value of both samples is 5807.7.

Both purified human insulin and the WHO standard human insulin are hydrolytically digested with protease V8. Fragments generated by hydrolysis are analyzed by ClS reverse phase HPLC. Both samples give an identical type of peptide mapping (see Thim et al., Genetics and Molecular Biology of Industrial Microorganisms, Hershberger et al., Ed. American Society for Microbiology, 1989, p. 322-328).

The present invention is not limited in scope by the deposited microorganism or the specific variants provided herein. In fact, various modifications of the invention in addition to those presented herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

Claims (16)

1. Chimeric protein, providing the formation of a bioactive conformation, containing the first peptidyl fragment with a length of 20-92 amino acids and having a sequence of at least 60% identical to the first 20 N-amino acid residues of SEQ ID NO: 1 or SEQ ID NO: 2, capable of binding to the antibody with human growth hormone and mediate the formation of a bioactive conformation of the peptidyl fragment of the precursor of human insulin; a peptidyl fragment of a human insulin precursor comprising human insulin chain A and human insulin chain B, wherein human insulin chain A and human insulin chain B are linked by a removable peptidyl fragment; wherein the C-terminus of the first peptidyl fragment is linked to the N-terminus of the peptidyl fragment of the human insulin precursor using Arg or Lys or by using a peptidyl binding fragment of at least two amino acids, the C-terminal residue of the peptidyl binding fragment being selected from the group consisting of from Arg or Lys. 2. The chimeric protein according to claim 1, characterized in that the peptidyl fragment of the insulin precursor contains the amino acid sequence of SEQ ID NO: 4. 3. The chimeric protein according to claim 1, characterized in that the peptidyl fragment of the insulin precursor contains the amino acid sequence of SEQ ID NO: 5. 4. The chimeric protein according to claim 1, characterized in that the first peptidyl fragment has a sequence identical to the amino acid sequence of SEQ ID NO: 1. 5. The chimeric protein according to claim 1, characterized in that the first peptidyl fragment has a sequence identical to the amino acid sequence of SEQ ID NO: 2. 6. The chimeric protein according to claim 1, characterized in that the peptidyl binding fragment consists of the amino acid sequence of SEQ ID NO: 3. 7. The chimeric protein according to claim 1, characterized in that the peptidyl fragment of the precursor of human insulin is able to bind to an antibody against human insulin. 8. The chimeric protein according to claim 1, characterized in that the peptidyl fragment of the precursor of human insulin has a sequence identical to the amino acid sequence of SEQ ID NO: 4. 9. The chimeric protein according to claim 1, characterized in that the deleted peptidyl binding fragment consists of 2-34 amino acids. 10. The chimeric protein according to claim 1, characterized in that the peptidyl fragment of the precursor of human insulin has the amino acid sequence of SEQ ID NO: 5. 11. The chimeric protein according to claim 1, consisting of the amino acid sequence of SEQ ID NO: 6. 12. The chimeric protein according to claim 1, consisting of the amino acid sequence of SEQ ID NO: 7. 13. The chimeric protein according to claim 1, characterized in that the first peptidyl fragment contains an amino acid sequence identical to the amino acid sequence consisting of residues 1-20 of SEQ ID NO: 1. 14. The chimeric protein according to claim 1, characterized in that the first peptidyl fragment contains amino acids with a higher charge than the fragment of the precursor of insulin; the positive charge of amino acid residues of the N-terminal portion of the first peptidyl fragment is higher than the negative charge of amino acid residues, and the negative charge of amino acid residues of the C-terminal portion is higher than the positive charge of amino acid residues, while the first peptidyl fragment contains an amino acid sequence of at least 60% identical to the amino acid sequence of SEQ ID NO: 1. 15. A method of producing a polypeptide or protein having insulin bioactivity, comprising expressing a chimeric protein according to claim 1; contacting the chimeric protein according to claim 1 with a chaotropic agent or with mercaptan, in which the peptidyl fragment of the insulin precursor acquires a conformation having insulin bioactivity, and purification of the protein with insulin activity. 16. A method for identifying a peptidyl fragment capable of mediating the formation of a bioactive conformation of an insulin precursor, comprising treating a chimeric protein according to claim 1, having a modified amino acid sequence of a first peptidyl fragment, and a chimeric protein according to claim 1, having an unmodified amino acid sequence of a first peptidyl fragment, measurement of the yields of a chimeric protein with bioactive conformation of insulin and comparison of the yield in the case of using modifiers peptide and unmodified peptidyl moieties.

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