Recombinant plasmid dna pet23-a(+)/prxvihumΔ178, encoding n-terminal fragment of human peroxiredoxine vi, and e.coli strain bl21/de3/pet23-a(+)/prxvihumΔ178 as producer of n-terminal fragment of human peroxiredoxine vi

FIELD: biotechnology, genetic engineering, pharmaceutical industry.

SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.

EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.

2 cl, 3 dwg, 4 ex

 

The invention relates to the field of biotechnology, genetic engineering and can be used to obtain the antioxidant drug peroxiredoxin designed for treatment of diseases associated with oxidative stress.

It is known that in case of incomplete reduction of molecular oxygen during cellular respiration formed reactive oxygen species - superoxide anion radical (), hydrogen peroxide (H2O2), hydroxyl radical (BUT·), which are extremely toxic to cells. Aerobic organisms have developed defense mechanisms to neutralize these substances. One of these protective mechanisms is the restoration of active forms of oxygen in the reactions catalyzed by enzymes - antioxidants. These proteins play an important role in maintaining the redox potential of cells. These include well-known antioxidants, such as superoxide dismutase, catalase, glutathione peroxidase, and, in addition, opened in the last decade peroxiredoxin [H.Z. Chae, K. Robison, L.B. Poole, Church, G., Storz, G., and Rhee S.G. (1994) Proc. Natl. Acad. Sci. USA, 91, 7017-7021].

Peroxiredoxin - a new family of proteins, which currently has more than 100 representatives found in all living organisms from archaebacteria to the man which thiol peroxidases [S.P. Lee, Hwang Y.S., Kim Y.J., Kwon for K.S., Kim H.J., Kim K., H.Z. Chae (2001) J. Biol. Chem., 276, 29826-29832].

In mammals identified 6 types of peroxiredoxins, differing in amino acid sequence, the mechanism of action and localization in the body and in the cage. All peroxiredoxin in their sequences contain highly conservative area, which is the active center of enzymes, which consists of one or two Cys residue. In tests in vitro have shown that peroxiredoxin prevent inactivation of glutamine synthase in the presence of Fe3+About2and dithiothreitol (DTT) - model oxidative system, generating free radicals [To Kim, I.H. Kim, K.Y. Lee, Rhee S.G., Stadtman E.R. (1988) J. Biol. Chem., 263, 4704-4711].

To date, 1-Cys peroxiredoxin (peroxiredoxin VI, PrxVI) identified in many organs and tissues of mammals. The first natural individual protein preparations PrxVI mammals were isolated from the olfactory epithelium [Peshenko I.V., Novoselov V.I., Evdokimov VA, Nikolaev Yu.V.. Shuvaeva T.M., Lipkin V.M., Fesenko E.E. (1996) FEBS Letters, 381, 12-14] and lung of rats [Kim T.S., Sundaresh C.G., Feinstein, S.I., C. Dodia, Skach W.R., M.R. Jain, Nagase T., Seki N.. Isherawa K., Nomura N., A.B. Fisher (1997) J. Biol. Chem., 272, 2542-2550]. These methods include the accumulation and tissue homogenization, extraction of the target protein, as well as thin fractionation of the drug using three consecutive chromatographic steps. Although tissue directly which edstone in contact with oxygen, the most enriched PrxVI [S.V. Novoselov, Peshenko I.V., Popov V.I., Novoselov V.I., Bystrova M.F., Evdokimov V.J., Kamzalov S.S., Merkulova M.I., Shuvaeva T.M., Lipkin V.M., Fesenko E.E. (1999) Cell Tissue Res., 298, 471-480], these labor-intensive and industrial reproducible methods have only theoretical value. The main disadvantages of obtaining PrxVI from natural sources are: the need for the accumulation of animal tissues, small final yield of pure drug (0.01 mg per animal) and the possibility of allergic reactions when using a foreign protein for treatment of humans.

Currently preparative number PrxVI mammals receive more preferred genetic engineering methods to produce the desired number of homogeneous genetic material (selected vector connected with the structural gene of the polypeptide) and, as a consequence, the final product is a protein.

So, in the cells of the strain E. coli BL21 (DE3) was performed biosynthesis of recombinant full-PrxVI person (PrxVIhum) [Chen L.-W., C. Dodia, Feinstein, S.I., Jain M.K., Fisher A.B. (2000) J. Biol. Chem., 275, 28421-28427]. This was taken a cDNA fragment PrxVIhum (PrxVIhum) HA0683 (GenBank™ D14662) length 1653 BP, containing an open reading frame for PrxVIhum (224 S.A.) size 672 BP a Large part of the original fragment (length 1044 BP) was built in expressing vector rats the restriction site HindIII. The resulting design to ensure ivala the accumulation of the recombinant protein, which, along with the amino acid sequence PrxVIhum contained 42 additional amino acid residue, including six His residues at the N-Terminus of the protein chain. Based on the same fragment-PrxVIhum and artificially typing sites for recognition of restricted NdeI and XhoI, the authors amplified the coding region. The resulting fragment was cloned on these sites in expressing vector 21b. In the recombinant protein biosynthesis which was determenirovana this plasmid contained only two additional amino acid residue, in addition to the six His residues at the C-end of the polypeptide chain of the product. After transformation of E. coli derived recombinant DNA and induction of gene expression by isopropylthioxanthone (IPTG) cells increased within 6 h and was destroyed; protein preparations were subjected to sequential chromatographic purification methods. The disadvantages of both of the products obtained can be attributed to the fact that, although the introduction of the polypeptide chain additional His residues greatly simplifies the selection of recombinant proteins, such modifications significantly shift the isoelectric point of the protein products compared to natural and, consequently, change their electrostatic microenvironment. In addition, the introduction of additional amino acid residues (42 in the first design the 2-x - the second increases the molecular weight of the product and, consequently, affects its penetration into the cell.

Expression of recombinant PrxVI was also carried out in the baculovirus system [T. Fujii, Fujii j, Taniguchi N. (2001) Eur. J. Biochem., 268, 218-224]. For this purpose from various rat tissues was isolated mixture of mRNA, which reverse polymerase reaction synthesized complementary DNA strand. Then this cDNA was subcloned into the baculovirus Shuttle vector pVL1392. The resulting design has provided a full-time PrxVI rats during infection of eukaryotic cells Sf21. Through planting, by fractionation on an ion-exchange resin with subsequent stages gel filtration functionally active recombinant protein was isolated from the culture fluid of these cells. The disadvantages of this method include the length (5 days) of the drug, the need to use expensive nutrient medium, low compared to bacterial systems, the yield of the target product and the possibility of adverse allergic reactions when used in pharmaceutical compositions rat PrxVI.

The closest in technical essence of the present invention is a polypeptide mass 25034 Yes, representing a full-size recombinant PrxVIhum and coding it recombinante the plasmid DNA pET23-a(+)/PrxVIhum [Merkulova M.I., Shuvayeva T.M., Radchenko V.V., Janine V.A., A.A. Cooper, Safin A.D., Lipkin V.M. (2002) Biochemistry, 67, 1496-1501]. Get peroxiredoxin has high antioxidant activity. However, high molecular weight, which prevents the penetration of molecules of antioxidant in the cells of the human body, limits its application.

The task of the invention is to construct plasmids determining the synthesis of truncated polypeptide PrxVIhum, preserving the antioxidant activity of the full-size PrxVIhum, as well as the creation of highly productive strain-producer to obtain a polypeptide peroxiredoxin man VI, which is the N-terminal fragment PrxVIhum.

The problem is solved by constructing recombinant plasmid DNA pET23-a(+)/PrxVIhumΔ178, encoding the N-terminal fragment peroxiredoxin VI person with a molecular mass of 19691,61 Yeah, containing RI-NdeI fragment of plasmid RET(+), including the promoter RNA polymerase of phage T7, the site of initiation of replication (ori) and the terminator of transcription of the ribosomal operon of E.coli, Ndel-EcoRl fragment of the gene PrxVIhum length of 552 BP, encoding PrxVIhumΔ178, genetic marker - Ar, determining the stability of the transformed plasmid RET-a(+)/PrxVIhumΔ178 cells of E. coli to ampicillin, a unique recognition sites of restriction endonucleases with shadowsinthegarden.com: NdeI-790, EcoRI-192, PvulI-1531, and also due to the strain E. coli BL21/DE3/pET23-a(+)/PrxVIhumΔ178 - producer of the N-terminal fragment peroxiredoxin VI person for synthesis of an N-terminal fragment PrxVIhum the size of 177 amino acid residues (PrxVIhumΔ178) with the expression level in 30% of the total cellular protein (30 mg/l of culture fluid).

An advantage of the claimed technical solution is the possibility of obtaining antioxidant peroxiredoxin VI of the person maintaining the antioxidant activity of the full-size peroxiredoxin at low molecular weight, which ensures penetration of the drug into the cells of the human body.

The starting plasmid for the construction of a new DNA sequence that encodes a polypeptide PrxVIhumΔ178 serves as a plasmid re-and(+)/PrxVIhum determining the expression of recombinant full-PrxVIhum. This plasmid construct a vector plasmid re-and(+)[Studier F.W., Moffatt, B.A. (1986) J. Mol. Biol., 189, 113-130]. Fragment PrxVIhum designed for cloning with preservation of the reading frame in expressing the vector obtained by polymerase chain reaction (PCR) [Taylor G. In: Polymerase Chain Reaction. A Practical Approach, v.1, M.J. McPherson, Quirke P., G. R. Taylor eds. Oxford Univ. Press. Oxford. 1994], using as primers oligonucleotides in a sequence which entered point replacement to create compliance is adequate restriction sites. As a direct use primer(underlined site recognition of restrictase NdeI), as a reverse -5'-CCATTAAGGCTGGGGTGTG-3' (underlined in the area of recognition of restrictase EcoRI). As a matrix for PCR using a plasmid containing the sequence PrxVIhum HA0683 (GenBank™ D 14662). The reaction mixture for PCR contains (in a volume of 50 μl): 1 ng of plasmid DNA, 20 pmol of each primer, 5 μl of buffer for PCR firm Promega, 200 μm of each dNTP, 5 units of Taq polymerase. The reaction starts with the pre-denaturation of the DNA - 94°C, 5 min followed by 30 cycles of PCR with the following parameters temperature cycle: denaturation 30 s at 94°S, annealing with primer - 30 s at 60°C, the elongation of 45 s at 72°followed by incubation at 72°within 5 minutes After treatment of the reaction product corresponding restrictase PrxVIhum clone in plasmid re-and(+) sites NdeI-EcoRI.

Recombinant plasmid DNA re-and(+)/PrxVIhumΔ178 characterized by the following features:

has the size 4210 BP

encodes N-terminal fragment PrxVIhum length 177 S.A.

consists of EcoRI-NdeI fragment of plasmid RET(+), including the promoter RNA polymerase of phage T7, the site of initiation of replication (ori) and the terminator of transcription of the ribosomal operon of E.coli, Ndel-EcoRI fragment lengths is th 552 BP sequence that encodes a PrxVIhumΔ178.

contains genetic marker - Ar, determining the stability of the transformed plasmid pET23-a(+)/PrxVIhumΔ178 cells of E. coli to ampicillin, as well as a unique recognition sites of restriction endonucleases, with the following coordinates: NdeI-790, EcoRI-192, PvuII-1531.

The advantages of the proposed design are achieved due to the fact that included in its membership a fragment PrxVIhumΔ178 encodes a shortened compared with PrxVIhum polypeptide that retains the antioxidant activity of the natural protein. This is, firstly, simplifies the chromatographic purification PrxVIhumΔ178; secondly, it makes more sophisticated use in the treatment compositions due to better permeability in the tissue and increase circulation time with biological fluids compared with the full-length protein; third, increases the proportion of the target product in the total biomass producer strain, which in turn leads to lower cost of the final product.

To obtain strain-producer of the polypeptide PrxVIhumΔ178 competent cells of E. coli BL21/DE3 transformed with recombinant plasmid DNA pET23-a(+)/PrxVIhumΔ178.

The resulting strain E. coli BL2/DE3/pET23-a(+)/PrxVIhumΔ178 characterized by the following features.

Morphological features: the cells are small rod-shaped, gram-negative, bore aronosky, 1×3.5 µm, motile.

Cultural characteristics: the growth on agar LB medium colonies are round, smooth, translucent, shiny, grey. The edge is smooth, the diameter of the colonies 1-3 mm thick, pasty consistency. Growth in liquid media (LB, minimal medium with glucose) is characterized by smooth turbidity, sediment easily sedimentary.

Physical and biochemical characteristics: the cells grow when 4-42°C, the optimum pH of 6.8 to 7.6. As the source of nitrogen used as mineral salts of nitrogen and organic compounds: amino acids, peptone, tripton, yeast extract. As a carbon source during growth on minimal medium using glycerol, carbohydrates, amino acids.

Resistance to antibiotics: cell producer strain are resistant to ampicillin (300 mg/ml), due to the presence of plasmid gene β-lactamase (bla).

Figure 1 presents the nucleotide sequence NdeI-RI-fragment of plasmid pET23-a(+)/PrxVIhumΔ178 and coded them amino acid sequence of the polypeptide PrxVIhumΔ178; figure 2 - physical map of the resulting plasmid; figure 3 - the results of a comparative study of protective properties of recombinant full-PrxVI person and its N-terminal fragment (PrxVIhumΔ178) for the protection of the glutamine synthase gene from inactivation in a model of oxidative system in vitro.

Example 1. Construction of recombinant plasmid DNA RET-a(+)/PrxVIhumΔ178, encoding the N-terminal fragment RGH VI person. Using the cDNA fragment RGH VI of the person who was previously cloned with preservation of the reading frame in expressing vector [Merkulova M.I., shuvayeva T.M., Radchenko V.V., Janine V.A., A.A. Cooper, Safin A.D., Lipkin V.M.(2002) Biochemistry, 67, 1496-1501]. This vector pET23-a(+)/PrxVJhum use as template for PCR. Thus obtained DNA fragment encodes N-terminal fragment RGH VI length of 177 amino acid residues. As a forward primer at this stage using 5'-GCG AAA TTA ATA CGA CTC ACT ATA GGG -3' (complementary to the promoter region of the vector pET23-a(+)/PrxVIhum). As a return for PrxVIΔ178 - 5'-CCA TCC TTCAAC TTA GGT GGC-3' (underlined site restrictase EcoRI allocated to the stop codon). The reaction mixture contains (in a volume of 50 μl): ~1 ng of plasmid DNA, 20 pmol of each primer, 5 μl of buffer for PCR (Promega, USA), 200 μm of each dNTP, 5 units of Taq polymerase. The reaction begins with preliminary denaturation of DNA at 94°C for 3 min followed by 10 cycles of PCR with the following parameters temperature cycle: denaturation 30 s at 94°S, annealing with primer - 30 s at 55°C, the elongation of 45 s at 72°then 10 cycles of reactions: denaturation 30 s at 94°S, annealing with primer - 30 with pri° With the elongation of 45 s at 72°followed by incubation at 72°within 5 minutes After processing the relevant restrictase fragment PrxVIhumΔ178 are ligated with NdeI-EcoRI fragment of plasmid re-and(+) using DNA ligase of phage T4. The accuracy of the Assembly, the check restriction analysis and sequencing of print paste according to the modified method of Singer [Chemeris AV, Akhunov ED, Vakhitov VA DNA Sequencing, M, “Science”, 1999]. Figure 2 presents the physical map of the recombinant plasmid pET23-a(+)/PrxVIhumΔ178.

Example 2. Expression PrxVIhumΔ178-fragment cDNA VI person. For expression of the fragment PrxVIhum as strain-owner choose the strain of E.coli BL-21(DE-3), bearing in chromosome gene RNA polymerase of phage T7 under the control of the inducible lac promoter [Studier F.W., Moffatt B.A. (1986) J. Mol. Biol., 189, 113-130]. Transformation of competent cells of E.coli BL-21(DE-3) carried out by a chemical method using calcium chloride [J. Sambrook, Fritsch e, Maniatis T. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press, N.-Y.]. For the recombinant protein, the cells are grown at 37°to advances in liquid culture, the absorption values And6000,6. Then for the induction of protein expression type inductor lac-promoter IPTG to a final concentration of 0.4 mm and continue incubation for another 5 hours After that, the cell suspension centrifuged. Sludge from the holding cells of the producer strain destroy ultrasound and re-centrifuged. The protein fraction containing the target product, are planted with saturated solution of (NH4)2SO4and cialiswhat against 12 mm Tris-Hcl buffer (pH 7,8), which consists of 1 mm MgCl2and 1 mm DDT. Protein blend chromatographic on DEAE-sepharose in a gradient of sodium chloride. The fractions containing the target polypeptide is subjected to further purification by gel-filtration on sephacryl S-200 and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Example 3. Comparison of the protective properties of recombinant full-PrxVIhum and its N-terminal fragment PrxVIhumΔ178 for the protection of glutamine synthase of E. coli from inactivation in a model of oxidative system in vitro.

Glutamylcysteine extracted from cells of E. coli strain DH5α [Streicher S.L., Tyier C. (1980) J. Bacteriol., 142, 69-78] and inactivate in the presence of Fe3+About2and DTT - in model oxidative system, generating free radicals [Kim K., Kim I.H., Lee K.Y., Rhee S.G., Stadtman E.R. (1988) J. Biol. Chem., 263, 4704-4711]. The reaction inactivation of glutamine synthase carried out in a volume of 60 µl reaction mixture containing 5 μg of enzyme, 50 mm Hepes (pH 7.4), 3 mm DTT, and 3 μm Fl3in the presence of different concentrations peroxiredoxin for 10 min at 37°C. Then determine the remaining active is th glutamine synthase of E. coli. Protective properties peroxiredoxin for the protection of glutamine synthase from E.coli inactivation is defined as the ratio of the remaining activity of the enzyme after inactivation in the presence of different concentrations peroxiredoxin to activity mainactivity glutamine synthase. The test results are presented in figure 3.

Example 4. Determination of productivity of the producer strain PrxVIhumΔ178.

To improve aeration in 5 ml liquid LB medium containing 100 μg/ml ampicillin, make individual colony of E. coli cells BL21/DE3 containing the constructed plasmid pET23-a(+)/PrxVIhumΔ178.

Grow at 37°on the rocking chair at 180 rpm for 2.5 h until in liquid culture, the absorption values A6000,6. Then add IPTG to a concentration of 0.4 mm and continue incubation in the same conditions for 6 hours Selected a sample of 1 ml and centrifuged 5 min at 6000 rpm, after which the cells are suspended in 100 μl of buffer containing 125 mm Tris-Hcl (pH 6.8), 20% glycerol, 3% sodium dodecyl sulfate and 0.01% bromophenol blue. The cell suspension is heated for 10 min in a boiling water bath. Selected samples of 2.5 µl 5 µl of 7.5 μl, 10 μl and 15 μl and analyzed by electrophoresis in 15%polyacrylamide gel containing 0.1% sodium dodecyl sulfate [Laemmli U.K. (1970) Nature, 227, 680-687]. Gel paint Kumasi R-250 and scanned on a laser densitometer Ultrascan XL. According to the data scanning polypeptide PrxVIhumΔ 178 accounted for 30% of total cellular protein, which corresponds to the output of pure protein product of 30 mg/l of cell culture.

1. Recombinant plasmid DNA pET23-a(+)/PrxVIhumΔ178 encoding the N-terminal fragment peroxiredoxin VI person, with a molecular mass of 19691,61 Yeah, containing EcoRI - NdeI fragment of plasmid RET(+), including the promoter RNA polymerase of phage T7, the site of initiation of replication (ori), genetic marker (Ar)determining the stability of the transformed plasmid RET-a(+)/PrxVIhumΔ178 cells of E. coli to ampicillin, a unique recognition sites of restriction endonucleases, with the following coordinates: NdeI-790, EcoRI-192, PvuII-1531, and NdeI - EcoRI-fragment gene PrxVIhum length of 552 BP, encoding PrxVIhumΔ178.

2. The strain E. coli BL21/DE3/pET23-a(+)/PrxVIhumΔ178 - producer of the N-terminal fragment peroxiredoxin VI person.



 

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