RU2235776C1 - Recombinant plasmid dna plp-3-1 encoding human polypeptide of proinsulin lyspro and strain of bacterium escherichia coli plp-3-1/tg-1 as producer of recombinant proinsulin lyspro - Google Patents

Abstract

FIELD: genetic engineering, molecular biology, medicine, endocrinology.

SUBSTANCE: invention relates, in particular, to preparing human proinsulin Lyspro and can be used for the development of medicinal preparations of new generation for treatment of insulin-dependent diabetes mellitus. Method involves construction of plasmid pLP-3-1 encoding hybrid polypeptide with the human proinsulin Lyspro sequence wherein sequence of domain B of staphylococcus protein A is connected through peptide linker HiS₆Gly/SerArg with amino acid sequence of human proinsulin of molecular mass 3.3 MDa (5051 base pairs) comprising Hind III/Bam HI-fragment of plasmid pPINS07 including hybrid tac-promoter, β-lactamase gene (bla), region of replication start (ori), E. coli transcription operator of ribosome operon, nucleotide sequence encoding the sequence of amino acids in domain B of protein B in S. aureus containing synonymous change C → T at position 3 of the third codon GAC connected with nucleotide sequence encoding peptide His₆GlySerArg, and Hind III/Bam HI-fragment (271 base pairs) encoding human proinsulin Lyspro, restriction sites with the following coordinates: Eco RI - 1, Nco I - 217, Bam HI - 221, Pst I - 342 and 417, Hind III - 492, Sal GI - 4513, Cla I - 4792. Microorganisms Escherichia coli are transformed with plasmid pPL-3-1 and the strain Escherichia coli TG-1/pLP-3-1 is obtained that is a producer of hybrid polypeptide with the human proinsulin Lyspro sequence. Invention allows preparing human proinsulin Lyspro with high yield by simplified technology.

EFFECT: valuable properties of plasmid and strain.

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Description

The invention relates to biotechnology, in particular to genetic engineering, in particular to the production of human Lyspro proinsulin, and can be used to create new-generation drugs for the treatment of insulin-dependent diabetes mellitus.

Strict control of the plasma glucose level in patients with diabetes is necessary to prevent severe complications of this disease. In healthy people, the plasma insulin level after a meal reaches a maximum after 30-40 minutes and slowly decreases over the next 2-3 hours. The onset of conventional insulin preparations after subcutaneous injection occurs slowly and its duration is much higher than the corresponding parameters characteristic of insulin formed in the body of healthy people after a meal rich in carbohydrates [Heinemann L., Starke AAR, Hohmann A., Berger M. // Horm. Metab. Res., 1992, v. 26 (Suppl), p.137-139]. When using conventional insulin preparations, there is a risk of early afternoon hyperglycemia, accompanied by hypoglycemia before the next meal. In this regard, there is a need for the creation of insulin preparations, which would be characterized by a faster onset and cessation of action after administration to the patient.

The Lyspro insulin analogue, in which at the positions 28 and 29 of the polypeptide chain there are Lys and Pro residues instead of Pro and Lys, respectively (Fig. 1), is the first high-speed insulin analogue, introduced into world clinical practice in 1996. Unlike natural insulin, Lyspro is characterized by a reduced ability to form aggregates and, as a result, faster absorption, a higher level of plasma content, as well as a shorter duration of action [Howey D.C., Bowsher R.R., Brunelle R.L., Woodworth J.R. // Diabetes, 1994, v. 43, p.396-402]. Such substitutions of amino acid residues do not affect the domain of the insulin molecule binding the receptor, and, as a result, the kinetic parameters of the interaction of the Lyspro insulin analog with receptors correspond to those of natural insulin. The action of Lyspro insulin begins 15 minutes after subcutaneous injection, reaches its maximum value after about 1 hour and stops after 2-4 hours [Torlone E., Fanelli C., Rambotti AM, Kassi G., Modarelli F., Di Vincenzo A., Epifano L., Ciofetta M., Pampanelli S., Brunetti P. // Diabetologia, 1994, v. 37, p. 713-720]. Currently, Lyspro insulin is widely used for the treatment of insulin-dependent diabetes in world clinical practice [Hanaire-Broutin N., Melki V., Bessieres-Lacombe S., Tauber J.P. // Diabetes Care, 2000, v.23, p.1232-1235; Jehle P.M., Aisenpreis U., Bundschu D., Keller F. // Fortschr. Med., 1999, v. 117, p.41-42].

A known method of producing insulin Lyspro, in which the nucleotide sequence encoding the insulin A chain was chemically synthesized and cloned in an expression plasmid vector under the control of the promoter of the E. coli tryptophan synthetase gene (trp LE ') as a hybrid gene comprising the 5'-terminal part β-galactosidase gene and the corresponding insulin sequence. The recombinant plasmid was introduced by transformation into a bacterial strain, where, after induction of the promoter, a hybrid protein of the following structure was synthesized: β-gal-Met-A chain (where β-gal is the N-terminal part of E. coli β-galactosidase, and Met methionine linker). The presence of a linker allowed cleavage of the chimeric polypeptide by cyanogen bromide in purified inclusion bodies, followed by chromatographic purification of the A chain. An insulin B chain containing the desired Lys (B28) -Pro (B29) sequence was obtained by solid phase synthesis. Then, the purified insulin chains were combined in vitro and the sulfhydryl groups were oxidized, which led to the formation of disulfide bonds between the chains. At the final stages, the Lyspro insulin thus obtained was purified [EP No. 0383472, MKI C 07 K 7/40, 1990].

The method has several disadvantages, in particular high complexity, low yield of the final product, which makes it impossible to use them for technological purposes.

The closest in technical essence to the present invention is a producer strain in which a human proinsulin gene containing two excess Met-Tyr amino acid residues at the N-terminus was cloned in an expression vector plasmid under the control of the pL promoter and temperature sensitive repressor cI857 phage λ. As a selectable marker, the tetRcycline resistance gene tetR was used. After thermal induction of the recombinant gene by increasing the temperature to 40 ° C, the resulting recombinant proinsulin accumulated in E. coli cells in inclusion bodies, from which it was further purified. At the first stage, using excess cathepsin C, Met-Tyr dipeptide was separated, at the second stage, the proinsulin C peptide was cleaved with trypsin and carboxypeptidase B, followed by purification of the formed Lyspro insulin analogue [US Pat. USA No. 5514646, MKI, 1996].

The need to use thermal induction to achieve controlled expression of Lyspro proinsulin in bacterial cells is a significant drawback of the discussed E. coli prototype strain. It is known that during the incubation of bacterial cells at elevated temperatures, the synthesis of heat shock proteins occurs, which also participate in the degradation of proteins with a disturbed spatial structure (which underwent incorrect post-translational folding or denatured during heat shock) [Gottesman S. // Annu. Rev. Gene., 1996, v. 30, p. 465-506; Baneyx F. // Curr. Opin. Biotechnol, 1999, v. 10, p. 411-421]. In addition, the high temperature at which bacterial cells were grown promotes aggregation of recombinant proteins in the cytoplasm [Clark E., De Bernardez // Curr. Opin. Biotechnol, 1998, v.9, p. 157-163]. All this leads to the formation of fragments of the polypeptide chain of proinsulin, enhances the aggregation of its molecules and reduces the yield of the native recombinant protein. Indeed, the yield of recombinant Lyspro proinsulin in the discussed prototype strain is ~ 2% of the total cellular protein.

The objective of the invention is the construction of a plasmid that determines the synthesis of a hybrid recombinant protein, in which a single IgG-binding domain of protein A is connected via the Hys ₆ AspGlyArg peptide linker to the amino acid sequence of human Lyspro proinsulin, that is, residues Lys and Pro located at positions 28 and 29 of the polypeptide chain, and the creation of a highly productive bacterial producer strain, allowing to obtain Lyspro insulin in high yield and by simplified technology.

The problem is solved by constructing a recombinant plasmid DNA pPL-3-1 encoding a hybrid polypeptide with the human Lyspro proinsulin sequence, in which the staphylococcal protein A domain B sequence is connected via the His ₆ GlySerArg peptide linker with the amino acid sequence of human Lyspro proinsulin, with a molecular weight of 3 , 3 MDa (5051 bp) containing the HindIII / BamHI fragment of the plasmid pPINS07, including the hybrid tac promoter, β-lactamase gene (bla), the region of origin of replication (ori), ribosomogen transcription terminator operon of E.coli, the nucleotide sequence encoding the amino acid sequence of domain B of protein A S. aureus, comprising substitution of synonymous C → T at position 3 of the third codon GAC, coupled to the nucleotide sequence encoding the peptide His ₆ GlySerArg, and a HindIII / BamHI fragment ( 271 bp) encoding human Lyspro proinsulin, restriction sites with the following coordinates: EcoRI - 1, NcoI - 217, BamHI - 221, PstI - 342 and 417, HindIII - 492, SalGI - 4513, ClaI - 4792, and strain Escherichia coli TG-1 / pLP-3-1 - producer of a hybrid polypeptide with the human Lyspro proinsulin sequence.

The starting plasmid for construction is the plasmid pPINS07, encoding a hybrid polypeptide consisting of one IgG-binding domain of Staphylococcus aureus protein A, peptide linker His ₆ GlySerArg and human proinsulin, hybrid tac promoter and transcription terminator of ribosomal operon E. coli RF1449 , MKI, publ. 2000]. As mentioned above, Lyspro insulin differs from ordinary insulin in the sequence of amino acids at positions 28 and 29 of its B chain. To introduce the corresponding mutation into the normal human proinsulin gene, a polymerase chain reaction (PCR) with overlapping primers is used. For this, first, the PCR products A and B (PCR 1, FIG. 2) are synthesized on the matrix of the initial plasmid using pairs of primers 1-4 and 2-3. Primers 3 and 4 contain non-complementary template nucleotides corresponding to the Lyspro sequence of the human proinsulin gene (indicated by asterisks). The resulting PCR products are electrophoretically purified on a DEAE membrane and used as a matrix in the second PCR reaction (PCR 2) in the presence of primers 1 and 2. The resulting PCR product C contains the desired mutation, as well as unique restriction sites of BamHI and HindIII on their ends. PCR product C is incubated with the restriction enzymes BATHI and HindIII and cloned in a pre-prepared vector, which is the original plasmid containing the recombinant gene of the hybrid polypeptide without the human proinsulin sequence deleted using the same restriction enzymes. The selection of recombinant clones is carried out by elimination of one of the restriction sites MbOII in the PCR product obtained by amplification of the corresponding sections of the recombinant plasmids using primers 1 and 2. The elimination of the restriction site is the result of a directed mutagenesis of the nucleotide sequence of the proinsulin gene of the original plasmid leading to the appearance in the B-chain of an altered sequence of amino acid residues Lys (B28) -Pro (B29) (figure 1). Plasmids of selected clones that meet the desired properties are purified by the alkaline method [Maniatis T., Fritsch EF, Sambrook J. // Molecular Cloning: a Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press], and the nucleotide sequence of the cloned DNA fragment is determined by the Sanger method using dideoxyribonucleotide derivatives as terminators of DNA synthesis with a thermosequenase and a fluorescent label, on an automatic sequencer. The resulting sequence is compared with the sequence of the original plasmid determined by the same method. The result of this work is to obtain a recombinant plasmid pLP-3-1 with the desired properties. Plasmid pLP-3-1 is introduced by electroporation into competent E. coli TG-1 cells, which leads to the creation of a recombinant strain of Lyspro hybrid proinsulin producer, E. coli pLP-3-1 / TG-1. A study of the expression level of Lyspro hybrid proinsulin in the obtained recombinant strain shows that the content of the recombinant polypeptide in bacterial cells after completion of induction with IPTG is at least 25% of the total cellular protein.

Recombinant plasmid DNA pLP-3-1 encoding a hybrid polypeptide with the amino acid sequence of the human Lyspro proinsulin analogue is characterized by the following features:

has a molecular weight of 3.3 MDa (5051 kbp);

encodes a fusion protein in which the staphylococcal protein A domain B sequence with the C-terminal hexahistidine linker is linked via the GlySerArg tripeptide to the human Lyspro proinsulin sequence;

consists of HindIII // BamHI fragment of plasmid pPINS07 containing a synthetic tac transcription promoter, β-lactamase gene (bla), which determines the resistance of bacterial cells to ampicillin, the region of plasmid DNA replication initiation (ori) ₁ transcription terminator of the E. coli ribosomal operon, a also comprising a part of the artificial gene encoding the IgG-binding domain of protein A from S. aureus, containing a synonymous C → T substitution in the third position of the third GAC codon of the 5'-terminal part, which improves the translation efficiency of the mRNA of the artificial gene by ribosomes, and xahistidine domain connected to the GlySerArg tripeptide, as well as BamHI // HindIII fragment encoding human Lyspro proinsulin, containing the LysPro sequence at positions 28-29 of its B-chain sequence, unique restriction sites have the following coordinates: EcoRI - 1, NcoI - 217 , BamHI - 221, PstI - 342 and 417, HindIII - 492, SalI - 4513, ClaI - 4792.

The advantage of the obtained plasmid pLP-3-1 over known plasmids is that it encodes the Lyspro proinsulin polypeptide under the control of a synthetic tac promoter induced by isopropyl-β-D-thiogalactopyranoside (IPTG). This allows you to induce and carry out the synthesis of proinsulin at a temperature of 37 ° C, which is usual for E. coli, which significantly increases the yield of the recombinant protein and facilitates its further purification. A further increase in the yield of recombinant Lyspro proinsulin was achieved by introducing a synonymous nucleotide substitution at the 5 ′ end of the expressed cassette of the recombinant nucleotide sequences. In addition, unlike the prototype plasmid, the constructed plasmid contains the β-lactamase gene as a selectable marker.

To obtain a producer strain of a hybrid polypeptide with human Lyspro proinsulin, the recombinant plasmid pLP-3-1 is introduced by electroporation into competent E. coli TG-1 cells.

The resulting strain of Escherichia coli / pLP-3-1, named E. coli LP31, is characterized by the following features.

Morphological signs: small rod-shaped cells, gram-negative, non-spore-bearing, 1 × 3.5 μm, motile.

Cultural traits: when growing on LB agar medium, the colonies are round, smooth, translucent, shiny, gray. The edge is even, the diameter of the colonies is 1-3 mm, the consistency is pasty. Growth in liquid media (LB, minimal medium with glucose) is characterized by even turbidity, the sediment easily sedimentes.

Physico-biochemical characteristics: cells grow at 4-42 ° C, optimum pH of 6.8-7.6. As a source of nitrogen, both ammonium mineral salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract. When growing on a minimal medium, glycerin, carbohydrates, amino acids are used as a carbon source.

Antibiotic resistance: cells of the producer strain show resistance to ampicillin (up to 300 μg / ml) and chloramphenicol (up to 100 μg / ml), due to the presence of resistance genes in the DNA of the recombinant plasmid pLP-3-1 and episomal DNA of the host strain, respectively.

The invention is illustrated by the following examples:

Example 1. Obtaining a DNA fragment encoding human Lyspro proinsulin.

100 ng of plasmid DNA pPINS07 containing the natural human proinsulin gene is used as a template for the synthesis of fragment A (Fig. 2) in the polymerase chain reaction (PCR), adding to 50 μl of the reaction mixture of the following composition: 20 mM Tris-Hcl, pH 8 8, 67 mM (NH ₄ ) ₂ SO ₄ , 1.5 mM MgCl ₂ , 0.2 mM dATP, dCTP, TTP and dGTP each, 0.05 units. Taq DNA polymerase and 1 μM of each of the primers CAGGATCATCACCATGGATCCCG and CACGACGGGTCGGCTTGGTGTAGAAG (primers 1 and 4, respectively, in figure 2, the mutated, mutagenizing sequence is underlined). Amplification of the corresponding plasmid region is carried out by conducting 22 PCR cycles (94 ° C, 20 s; 50 ° C, 20 s; 72 ° C, 20 s). The synthesis of fragment B (figure 2) is carried out under the same conditions and in the same reaction mixture, but in the presence of primers CCGCCAAGCTTACTAGTTGCAGTAG and ACACCAAGCCGACCCGTCGTGAAGC (primers 2 and 3, figure 2). The synthesized fragments A and B were electrophoretically transferred in 3% agarose gel onto pieces of a NA-45 DEAE membrane (Schleicher & Schuhle) and eluted in centrifuge microtubes in 1.5 ml of 200 μl buffer containing 1.5 M NaCl, 10 mM EDTA for 40 min at 65 ° C. E. coli tRNA is added to the buffer to a final concentration of 100 μg / ml, and the synthesized fragments are precipitated with 2.5 volumes of 96% ethanol. The precipitate was collected by centrifugation on an Eppendorf microcentrifuge at maximum speed for 15 minutes, the supernatant was discarded, the precipitates were washed with 1 ml of cold 70% ethanol, dried at room temperature and dissolved in 50 μl of deionized water. Assembly of the complete fragment C (Fig. 2) is carried out by PCR in 25 μl of the above reaction mixture, however, in contrast to it, containing 1 μl of purified overlapping fragments A and B as a matrix, as well as primers CAGGATCATCACCATGGATCCCG and CCGCCAAGCTTACTAGTTGCAGTAG (primers 1 and 2 figure 2). To obtain sticky ends, the synthesized fragment C is incubated with restriction endonucleases HindIII and BamHI (Fermentas, Latvia). At the end of the incubation, fragment C containing sticky ends is precipitated with 96% ethanol, as described above, but without adding tRNA, washed with 70% alcohol, dried and dissolved in 20 μl of water.

Example 2. Preparation of an expression vector for cloning a synthesized fragment C.

3 μg of plasmid pPINS07 was incubated with HindIII and BamHI restriction endonucleases as described above, and the vector DNA fragment further used to construct the expression plasmid was purified electrophoretically using low-melting agarose. For this, the fragments formed after restriction are separated by electrophoresis in 1.5% agarose, the desired fragment is transferred by electrophoresis to a well filled with low-melting agarose, and isolated from agarose by the phenolic method [Maniatis T., Fritsch EF, Sambrook J. // Molecular Cloning: a Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press]. The fragment is precipitated and washed with alcohol, as described above, in the presence of tRNA as a carrier and dissolved in 20 μl of deionized water.

Example 3. Obtaining a plasmid pLP-3-1 expressing the human Lyspro hybrid proinsulin gene and the Lyspro hybrid proinsulin producing strain.

10 μg of a large fragment of plasmid pPINS07 containing the sticky ends of HindIII / BamHI are ligated with 50 μg of purified fragment C with the same sticky ends [Maniatis T., Fritsch EF, Sambrook J. // Molecular Cloning: a Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press]. The reaction products are precipitated and washed with ethyl alcohol, as described above, and dissolved in 10 μl of water; 5 μl of ligation products are used to transform competent E. coli TG-1 cells by electroporation under standard conditions. A suspension of transformed cells (200 μl) was plated on LB agar containing ampicillin (100 μg / ml) as a selection agent. Among the grown bacterial colonies, PCR in the presence of primers 1 and 2 (FIG. 2) was used to select clones containing an insert of the cloned fragment C. For this, the resulting PCR products were incubated with the restriction endonuclease MboII (Fermentas, Latvia) and the resulting DNA fragments were analyzed by electrophoresis in 5 % agarose. If there is a Lyspro mutation in the analyzed plasmid DNA as a result of restriction, only three DNA fragments are formed, since one of the three restriction sites (central, Fig. 2) contained in this part of the original plasmid pPINS07 is eliminated by the introduced mutation. Plasmid DNA was isolated from the clones containing the desired mutation by the standard phenolic method [Maniatis T., Fritsch EF, Sambrook J. // Molecular Cloning: a Laboratory Manual, 1982, Cold Spring Harbor Laboratory Press] and the nucleotide sequence of the Lyspro proinsulin gene was determined by the method Sanger on an automatic sequencer (Amersham & Pharmacia) using fluorescently labeled primers. At the final stage of work, among the clones containing the Lyspro proinsulin gene, a spontaneously generated mutant E. coli clone pLP31 is selected, the plasmid DNA of which contains a synonymous C → T substitution at position 3 of the third GAC codon of the 5 ′ terminal sequence of the artificial gene encoding the IgG binding domain S. Aureus protein A, which, without changing the amino acid sequence of the hybrid polypeptide, leads to an increase in its expression in bacterial cells, possibly due to an increase in the stability of its mRNA.

Example 4. The determination of the productivity of the producer strain of the hybrid polypeptide with human Lyspro proinsulin.

An individual colony of E. coli pLP-3-1 / TG-1 cells containing the plasmid pLP-3-1 is added to 20 ml of LB liquid medium containing 100 μg / ml ampicillin and grown at 37 ° С on a shaker at 180 r / min for 4 hours to a turbidity of 0.8. Then, an IPTG inducer was added to a final concentration of 0.5 mM and incubation was continued under the same conditions for 6 h. A 2 ml sample was taken and centrifuged for 5 min at 6000 rpm, after which the cells were suspended in 200 μl of buffer containing 125 mM Tris -HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol and 0.01% bromophenol blue, heated for 10 min in a boiling water bath. Samples of 2.5, 5, 7.5, 10 and 15 μl were taken and analyzed by electrophoresis in a 13% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The gel is stained with Coomassie R-250 and scanned on an Ultrascan XL laser densitometer. According to the scan, the hybrid polypeptide makes up at least 25-30% of the total cellular protein.

Claims (2)

1. Recombinant plasmid DNA pPL-3-1 encoding a hybrid polypeptide with the human Lyspro proinsulin sequence, in which the staphylococcal protein A domain B sequence is connected via the His ₆ GlySerArg peptide linker with the amino acid sequence of human Lyspro proinsulin, with a molecular weight of 3.3 MDa ( 5051 bp) containing the HindIII / BamHI fragment of the plasmid pPINS07, including the hybrid tac promoter, the b-lactamase gene (bla), the region of origin of replication (ori), the transcription terminator of the E. coli ribosomal operon, the nucleotide sequence encoding conductive domain amino acid sequence in protein A S. aureus, comprising substitution of synonymous C → T at position 3 of the third codon GAC, coupled to the nucleotide sequence encoding the peptide His ₆ GlySerArg, and the HindIII / BamHI / fragment (271 bp) encoding human Lyspro proinsulin, restriction sites with the following coordinates: EcoRI-I, NcoI-217, BamHI-221, PstI-342 and 417, HindIII-492, SalGI-4513, ClaI-4792. 2. The strain Escherichia coli TG-1 / pLP-3 -1-producer of a hybrid polypeptide with the human Lyspro proinsulin sequence.

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