Recombinant plasmid dna encoding the biosynthesis of human alpha 2-interferon, and method thereof

 

The invention relates to the field of genetic engineering and biotechnology. By insertion into the vector pQE30 gene2-interferon and inverted htpR gene constructed recombinant plasmid DNA pASIF62. Also the method of biosynthesis2-interferon in E. coli cells, followed by purification of the desired product, namely, that transform E. coli plasmid DNA pASIF62, and cultivation is carried out to achieve a turbidity of 0.4-0.6 ML, after which the medium was added to 0.1-0.3 mm isopropyl-b-thiogalactoside. The use of the invention provides an increased output2-interferon. 2 S. and 2 C.p. f-crystals, 8 ill., 1 PL.

The invention relates to genetic engineering, specifically genetic design, coding synthesis2-interferon person, and which can be used for industrial production in E. coli.

Interferons were first attracted attention due to its ability to interfere with the replication and production of various kinds of viruses. This family of multifunctional cytokines, among which the most studied are interferon-alpha (IFN-represents a whole family of proteins which are encoded by at least 23 different genes. This 19-23 kDa polypeptides ranging in length from 156 to 172 amino acids. All IFN-vysokoekonomichny and differ only in one or two amino acids. In a protein sequence are potential sites of glycosylation, but most of the subtypes of IFN-deglycosylation. Spatial structure of interferon consists exclusively of alpha-helices and is classified as "chetyrehspalnyh barrel". Biological activity have monomers of this protein.

Interferons have many activities that contribute to the activation of T-cell immunity, which is necessary for the natural protection of the body against viral and bacterial infections. These cytokines, aktiverat other immune cells - T-cells, NK-cells, activated macrophages, stimulating early non-specific immune response. They enhance the expression of receptors and antigens to effector cells, regulate the transcription of new genes that stimulate the expression of proteins of the major histocompatibility complex class II (MHC II). Interferons find antiproliferative, the t part in the regulation of haematopoiesis, resulting in enhanced production and activation of neutrophils, monocytes, platelets, which, in turn, is a key event of the inflammatory process. And this is only a small part of the activities possessed by interferons.

In sum, we can conclude that the use of exogenous interferon for the treatment of many diseases - inflammatory, neoplastic, and many others, where necessary immunomodulating activity, more than justified. In connection with the above-described properties of interferon many researchers have developed strains-producers for the recombinant analogue of this protein (patent WO 01/42301 A1, WO 01/25438 A2, WO 00/69913, WO 01/57217 A1, WO 87/06616, WO 88/09673, EP 0032134 B1, EP 0626448 A2, EP 0538300 B1, US 5710027, SU 1092176 A).

Closest to the technical essence is recombinant DNA, its producing strains on the basis of E. Li, which is suitable for industrial production of2-interferon (US 5710027, kN. 435-69.51, 1998).

Recombinant DNA contains the nucleotide sequence,2-interferon gene, fused with a nucleotide sequence of the signal peptide heat-resistant enterotoxin E. coli, under the control of the E. Li phosphatases promoter (phoA protein in periplasmatic space cells and gives well-formed with authentic N-termination end and the correct disulfide bonds2-interferon (i.e. biologically active2-interferon) with suitable industrial output - 15-20% of total cellular protein.

However, the known recombinant DNA vector containing it, and used the strain give the product, which requires a multi-stage purification, including sequential chromatography on silica gel, chromatography with hydrophobic interaction, cation-exchange and anion-exchange chromatography.

The inventive objective is to increase the yield and simplify purification of biologically active (i.e. with preserved structural features noted above)2-interferon.

Inventive task is solved in that the proposed recombinant plasmid DNA pASIF-62 encoding synthesis2-interferon, which has a size of 4200, etc., of O., and consists of the following elements: lac-promoter, the ATG codon, gene2-interferon size 642 nucleotide, inverted DNA fragment-htpR gene size 350 nucleotides, gene resistance to ampicillin and sites for restricted Pst, Hind III, BamHI, and contains 18 nucleotide residues, operatively associated with the lac-promoter and the gene of interferon and coding herona the expression of its gene in the composition of recombinant DNA in E. Li, the difference is that the expression is carried out in a recombinant plasmid pASIF-62 in the growth medium up to its turbidity of 0.4-0.6 ML, after which it add 0.1-0.3 mm isopropyl-b-thiogalactoside - inductor l-promoter (IPTG) and expression continue under the control of induced lac promoter in recombinant plasmids. Purification of the target product, obtained in plasmid DNA pASIF-62 is carried out in one stage by the method of cation-exchange chromatography on agarose media. In the method, it is assumed as the preferred option to use strains of E. Li, defective gene htpR.

In the proposed method the expression of the interferon is in the cytoplasmic environment of E. Li, which creates certain difficulties in the correct folding of the protein, as well as in the selection of the leader of the stable expression, which should not change the authenticity of the termination end.

Another problem cytoplasmic expression of heterologous proteins in E. coli is the problem of multi-stage purification, requiring expensive in the first stage of purification using immunoaffinity chromatography (EP 396555).

The proposed recombinant plasmid to obtain2-interferon and the method for its AI proving “inventive step” of the proposed invention.

It is known that the aggregation alien recombinant polypeptides in E. coli cells respond “chaperones”, i.e. proteins of the heat shock (HSP). These include proteins DNAK, groES and chaperonin, which cause aggregation of non-native polypeptides, and Lon-protease exercising their electoral degradation. Regulation of transcription of genes encoding proteins of the heat shock protein is htpR, which is the alternative-subunit RNA polymerase and when it was embedded in groverment provides selective transcription of HSP genes in the conditions of synthesis of abnormal proteins. Previously we established that in the cells of E. coli defective in HSP, there is more intensive accumulation of recombinant proteins, and Taurus enable obtained in these strains was significantly higher yield proteins with natural conformation (with the correct folding).

These observations allow us to conclude that a violation of the functions of chaperones in E. coli cells makes the cells very useful from a biotechnological point of view of properties, is considerably increased “output” recombinant products.

However, the known mutations that disrupt the function of chaperones and reduce proteolytic activeseries cultivation of recombinant strains.

We proposed another solution to this problem, consisting in the fact that the recombinant plasmid encoding synthesis2-interferon, for a sequence of IFN gene contains a short inverted sequence htpR gene-regulator of HSP gene expression. The RNA molecule formed during the transcription of this sequence, have a structure complementary to the structure of the mRNA of the gene htpR and can perform the function of antisense RNA, blocking the synthesis of HSP by inactivation of protein HtpR.

Made by the Applicant the choice of the leader sequence of six his-tag residues corresponds to the greatest degree major biotech claims that may be presented to him.

It is, first and foremost, a reliable expression merged with him2-interferon, the lack of appropriate in such cases, point substitutions to adapt to belauxinteziruushih apparatus of the host cell, simplifying the final isolation and purification of fused protein, as well as the absence of obstacles for the correct folding of the Mature protein.

To obtain the recombinant DNA was used vector pQE30 containing the lac promoter, the site of translation initiation, the area code is blowing examples.

Example 1.

Cloning of the gene2-interferon person.

As a source of genes2-interferon were used:

1. Library of human genes

2. Plasmid from industrial producer strain2-interferon based on Pseudomonas Putida

3. Plasmid from industrial producer strain2-interferon-based Escherichia coli.

A fragment of the gene IFN2 corresponding to the Mature part of the protein, was isolated from three of the above sources method of amplification using the primers:

IFNA2-L gc gga tcc atg tgt gat ctg cct caa ace

IFNA2-R ccc aag ctt tea ttc ctt act tct taa.

The fragment was treated with restrictase BamHI+HindIII and cloned between the BamHI and HindIII by Sagami three types of vectors for the expression.

In each case obtained and analyzed on 20 independent clones.

Induction of interferon synthesis was assessed in 10 ml of IPTG-induced cultures (growing up to OD600=0.5, introduction to 0.2 mm IPTG, and then 3 hours at C on the rocking chair). The protein products of the expected size checked by SDS-PAGE.

As cells“owners” were used E. coli strains BL21, DLT1270, DSK-41. Strain DSK-41 obtained in the laboratory of molecular biology WINCMD by making mutations in the gene htp is different proteins. Another feature of the strain DSK-41 is that in the stationary growth phase in this strain is observed spontaneous derepressed genes under the control of the lac promoter and lac-repressor, probably due to degradation of the lac-repressor.

Primers:

IFNA2-L gc gga tcc atg tgt gat ctg cct caa ACC

IFNA2-R ccc aag ctt tca ttc ctt act tct taa

In Fig.1 shows the sequence of the gene for human IFNA2, where the beginning of the gene - 69 HT (ATG), the end - 635 HT (TGA), the Mature portion of the protein corresponds to the area 138-635 NT. The beginning of the gene, the beginning of the Mature part and the stop codon are underlined. For expression taken Mature part. Was inserted between the BamHI/HindIII sites of pQE30.

Example 2.

Cloning of the gene sequence htpR.

A fragment of the htpR gene was isolated from plasmid pKV3 containing polymerase htpR gene from the genome of E. coli. Plasmid DNA was treated with restrictase Hind III and Pst I and after fractionation on an agarose gel were isolated fragment of the htpR gene size 350 base pairs. Fig.2 is a hypothetical scheme of interaction of the mRNA htpR gene with antisense RNA.

Example 3

The study of the proteolytic activity of the strains of E. coli containing plasmid pASIF62.

To construct the plasmid, the coding synthesis2-interferon, used the vector pQE30 where Stralis 6 histidine residues. Then in the 3’-terminal region of the gene after termination codons were embedded fragment htpR gene in the reverse orientation, as described in example 2. The General scheme of the genetic structure shown in Fig.3 (a Recombinant plasmid pASIF62).

It is assumed that the induction of interferon synthesis in E. coli cells in the presence of IPTG will produce antisense transcript inverted fragment htpR gene, which when interacting with mRNA htpR will block the synthesis of HSP and, therefore, reduce the level of proteolysis in E. coli cells.

Determination of proteolytic activity of strains

The rate of proteolytic degradation of proteins was measured according to the method of Goldberg. Bacterial cells were grown at 30°C in minimal medium M9 containing the necessary additives. Upon reaching an optical density of the culture at 550 nm = 0.4 to 0.5 in the environment contributed puromycin to a final concentration of 300 μg/ml after 15 min L3H-leucine to a final concentration of 0.2 CI/ml After the cells were grown for 10 min, collected by centrifugation, washed twice in M9 medium containing cold leucine at a concentration of 75 μg/ml to prevent reclosing3H-leucine released from the material puromycin polypeptides was determined by the accumulation of labeled free amino acids in THU-soluble fraction. As follows from the results presented in Fig.4A and 4B (the rate of degradation3H-puromycin polypeptides in strains:-BL21c the plasmid pASIF62,-BL21 without plasmids pASIF62) - in strain BL21 E. Li, not containing plasmid pASIF62, there has been a significant increase in proteolysis at a temperature of +42°C, while in the strain containing plasmid, the level of proteolysis in similar conditions is significantly lower.

Example 4

Synthesis2-human interferon in E. coli

The level synthesis2-interferon was determined in strains of E. coli containing plasmid pIF62 (not containing the htpR gene sequence and having a normal level of proteolysis) and plasmid pASIF62, inhibitory synthesis of proteases.

E. coli cells were grown to a density of OD-05, made IPTG to a concentration of 0.2 mm for induction of interferon synthesis. Cellular proteins were fractionally 13% SDS-Page.

From the data shown in Fig.5, it follows that in identical growth conditions E. coli cells containing plasmid pASIF62, synthesize recombinant2-interferon. In Fig.5 shows the electrophoresis of proteins of E. coli in polyacrylamide gel, where

1 - markers moehren,

3 - strain of E. coli with the recombinant plasmid.

Example 5

Selection of recombinant2-interferon

Cells of E. coli strain DLT1270 transformed with plasmid pASIF62 and grown over night at 37°C in LB medium with the addition of ampicillin (100 μg/ml) and glucose (0.2 percent). Then was diluted overnight culture 25 times with fresh LB medium with ampicillin and pokasivali 2-3 hours until the turbidity of about 0.5, after which the solution was added IPTG to 0.2 mm. Raised another 3 hours, centrifuged culture with 5 thousand rpm for 10 min, the supernatant was discarded.

The precipitate after centrifugation suspended in buffer 20 mm Tris-Hcl, pH 8, 0.3 M NaCl (1 ml precipitated from 50 ml culture) and was voiced by 6 pulses for 15 seconds. Centrifuged 10 thousand rpm, 10 min, the precipitate was washed with the same buffer with the addition of 0.1% Triton X-100 (0.5 ml), then suspended using a homogenizer in 5 ml of buffer A (10 mm Tris-Hcl, pH 8, 6 M urea, 0.4 M NaCl, 10 mm p-mercaptoethanol, 5 mm imidazole). Was stirred for 1 hour at room temperature, separated supernatant by centrifugation at 10 thousand rpm for 10 min, the precipitate was discarded.

Column ID-agarose (1 ml) was charged with a solution with NISO4, balanced with buffer And 5 mm imidazole. The supernatant was applied to a column, The h 8, 2M urea, 0.4 M NaCl, 10 mm p-mercaptoethanol, 0.3 M imidazol). This buffer was added to 0.5 ml, collecting the appropriate fractions. Usually squirrels HI 6 HI and 8 were found in fractions 1-3.

Combined fractions were dialyzed overnight against 100-fold volume of buffer 10 mm Tris-HCl, pH 8, 150 mM NaCl.

Selected2-interferon was characterized by liquid chromatography and electrophoresis Page. In Fig.6 presents data electrophoretic analysis of recombinant2-interferon in polyacrylamide gel, where M is the marker; INF2-interferon.

In Fig.7 presents chromatographic characterization of recombinant2-interferon person.

The invention is illustrated by table.

The output amounted to 30% by weight of the total cellular protein.

As follows from the data presented, described the method of allocation allows to obtain a homogeneous protein, devoid of additional impurities.

Example 6

Immunochemical characterization of recombinant2-interferon.

To identify recombinant protein were held �232D/chr/945.gif">2-interferon person. As a standard was used commercial drug2-interferon (intron), manufactured by Schering-plough (USA). In Fig.8 shows the results of experiments confirming the complete identity of the two proteins.

Claims

1. Recombinant plasmid DNA pASIF62 encoding the biosynthesis of the human2-interferon, which is characterized by the fact that it has a size of 4200, etc., of O., and is a vector Q30 with lac-promoter, a resistance gene for ampicillin and sequence of 18 nucleotides that encodes a 6 his-tag residues, which promptly incorporated gene interferon size 642 nucleotides (between sites for restricted BamHI and HindIII) and inverted DNA fragment htpR gene size 350 nucleotides (between sites for restricted HindIII and > PST).

2. The method of obtaining human2-interferon by the expression of its gene in the composition of the recombinant vector in E. coli, followed by purification of the target protein, wherein the gene expression is carried out in the composition of the recombinant plasmid DNA pASIF62, E. coli cells are cultivated until the turbidity of 0.4-0.6 ML, after which credet.

3. The method according to p. 2, characterized in that the use of mutant gene htpR strains of E. coli.

4. The method according to p. 2, characterized in that the purification of the target product is carried out in one stage by the method of cation-exchange chromatography on agarose media.

 

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