Recombinant bifunctional psh protein possessing antioxidant activity of superoxide dismutase and peroxidase, chimeric nucleic acid coding it, recombinant plasmid vector containing it and using psh protein in reperfusion injury of heart

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and represents a new bifunctional PSH protein containing human peroxiredoxin Prx6 and manganese superoxide dismutase MnSOD possessing the antioxidant activity of superoxide dismutase and peroxidase. What is also described is a chimeric nucleic acid coding the presented protein. A method for preparing the presented protein by culturing cells of the strain E.coli BL21(PSH) transformed by constructed recombinant expression vector based on pET22b(+) plasmid is disclosed.

EFFECT: invention enables producing high-yield protein PSH possessing the high antioxidant activity.

4 cl, 6 dwg, 3 tbl, 7 ex

 

The scope of the invention

The invention relates to the field of biotechnology, genetic engineering and can be used to obtain the antioxidant drug.

The level of technology

Currently, it is widely accepted that acute pathological processes in humans are accompanied by oxidative stress in various tissues of the body (the hyperproduction of reactive oxygen species), which is one of the major damaging factors in the development of acute inflammatory processes.

A wide range of diseases, the pathogenesis of which plays an important role in oxidative stress, forced to seek ways to effectively neutralize reactive oxygen species (ROS), as in the majority of cases, the situation can be significantly adjusted using antioxidant drugs. In medicine it is widely used medicines antioxidant action of both natural and synthetic origin. However, in most cases, they are relatively inefficient and are used as additional medicines or as a preventive tool. This fact has stimulated research fundamentally new drugs with antioxidant action. One promising direction could be the use of enzymes-antioxidants as the basis for drugs with antioxidant action, because the effectiveness of these enzymes by several orders of magnitude higher than the antioxidant activity of commonly used low molecular weight compounds.

To date, there are already experimental evidence suggesting that the enzymes-antioxidants, primarily peroxiredoxin and superoxide dismutase, may be promising for use as a main component drugs of antioxidant action in the treatment of pulmonary and cardiovascular diseases and in transplantation and conservation bodies.

There are several representatives of the recombinant proteins used as antioxidant agents in compositions for the treatment of mammals. One of the representatives of these proteins is peroxiredoxin PrxVI.

Known: recombinant plasmid DNA-based expression plasmids pet-23a, an Escherichia coli strain transformed with this recombinant plasmid DNA, producing the recombinant protein peroxiredoxin PrxVI [patent RU No. 2280448, class AC 31/38, AK 38/05, publ. of 10.05.2004]. Known application of fragment peroxiredoxin PrxVI, with high efficiency in compositions for treating mammals with the smaller sizes of the protein [U.S. patent No. 8003345, Fesenko, et al. Antioxidant pharmaceutical compound, method for producing polypeptide and method of cure, publ. 2.08.2011].

It is known the use of pharmaceutical compositions for preventing and treating restenosis, comprising as an active ingredient is protein peroxiredoxin prx2 decryption or gene construct for expression in the cells of the body [patent US 7794708, Kang, et al. Pharmaceutical composition for preventing and treating restenosis comprising peroxiredoxin 2 protein as an active ingredient and a composition for screening therapeutics of restenosis comprising peroxiredoxin 2 gene or its protein product, and method for screening therapeutics ofrestinosis using said composition, 14.09.2010].

There is a method of treating heart disease by direct injection into the heart of a vector containing a nucleotide sequence encoding a protein AOP-1 (similar to protein PrxVI), to improve the production of the protein AOP-1 in the heart, protect the heart from damage and maintain the pulse [US patent 7598228, Hattori et al. Therapeutic methods and agents for diseases associated with decreased expression of AOP-1 gene or AOP-1, 06.10.2009].

Another direction in creating an effective antioxidant agents refers to the attempts to create antioxidants consisting, for example, of two different types of proteins.

Known examples of the formation of fused proteins, which proteins or their domains, for example, responsible for transport into the cell, were covalently linked to target proteins, which were Cu/Zn superoxide dismutase (SOD) [U.S. patent No. 7306944, Choi, S. Park, J. Advanced cell-transducing transport domain-target protein-transport domain fusion protein and uses thereof, 2007-12-11].

Known primaryprimary bifunctional recombinant proteins in mammals, which includes the kinase and endonuclease [U.S. patent No. 7439061, Kaufman et al. DNA encoding the novel mammalian protein, Irelp, 21.10.2008].

Closest to the technical solution of the present invention is the use of antioxidant therapy using a combination of SOD and catalase (CAT) in the experiments to create covalently linked biofermenter conjugate SOD-chondroitin sulfate - CAT. [Maksimenko AV, Golubykh VL, Tischenko EG. The combination of modified antioxidant enzymes for anti-thrombotic protection of the vascular wall: the significance ofcovalent connection ofsuperoxide dismutase and catalase activities. J Pharmacy Pharmacol 2004; 56: 1463-8., A. Century Wavev, A. C. Maksimenko. Enzymatic antioxidants on the way to practical medicine http://www.consilium-medicum.com/article/18782)]. To obtain biofermenter conjugate, SOD-1 was covalently attached through glycosaminoglycan vascular wall - chondroitin sulfate (CS) - the CAT and the obtained adduct of SOD-CHS-CAT. Product superoxiddismutase reaction, hydrogen peroxide was found to be a substrate for subsequent CAT transformation in a safe in these conditions, water and molecular oxygen. However, these authors as peroxidase was taken catalase, which significantly limited range natalizumab of hydroperoxides (destroyed only hydrogen peroxide). The process of creating chimeric bifunctional enzyme was carried out chemical curing commercially available farm is tov, that led to a significant decrease in the enzymatic activity of each component of the chimeric protein. The last fact is associated with the lack of selectivity in chemical linking of proteins, which may shield a significant portion of the active centers of enzymes. However, it was shown significant therapeutic effect of this chimeric protein in protecting organs from oxidative stress caused by ischemia-reperfusion. It is shown that bifermentans conjugate proved effective antithrombotic agent actions.

One of the objectives of the present invention is to eliminate these disadvantages of chemical cross-linking of enzymes by constructing chimeric nucleic acid that encodes a bifunctional protein containing the amino acid sequence peroxiredoxin person (Prx6) and amino acid sequence of Mn-superoxide dismutase (SOD). When this protein retains high antioxidant activity as Prx6, and Mn-SOD.

Another object of the present invention is to provide a highly productive strain-producer to obtain bifunctional polypeptide PSH.

The next task is the use of bifunctional polypeptide in medicine, in particular in compositions used for reperfusion of the heart.

Put what the tasks are solved by creating a bifunctional enzyme antioxidant PSH (Peroxiredoxin-Superoxiddismutase-GIS-tag), which is able to neutralize all basic types of reactive oxygen species (superoxide radicals, inorganic and organic hydroperoxides and peroxynitrite).

In the beginning creates a gene construct encoding both consistently protein, then it is included in expressing vector RET(DE), on the basis of which creates a strain of E. coli pET23(PSH), which provides a synthesis of the polypeptide PSH

The invention

One of the objects of the invention is a chimeric nucleic acid encoding a hybrid protein PSH, including human peroxiredoxin Prx6 and the manganese superoxide dismutase MnSOD, possessing antioxidant activity of superoxide dismutase and peroxidase, primary structure which describes the amino acid sequence SEQ ID NO:1.

Another object of the invention is a recombinant expression vector pET23(PSH)containing the chimeric nucleic acid encoding a protein PSH with the amino acid sequence SEQ ID NO:1, operatively integrated into a plasmid pET22b(+) restriction sites NdeI and XhoI with C-terminal localization polyangitis.

The next object of the invention is a hybrid protein PSH for reperfusion of the heart, the primary structure of which is described amino acid sequence of SEQ ID NO:1.

List of figures

Fig.1 Obtaining the plasmid pET23(PSH). Where a is the gaining the genes peroxiredoxin and superoxide dismutase; In the stitching genes PCR with primers per1 and sod2; With the PCR restriction fragment enzymes NdeI and XhoI; D - embedding treated PCR fragment in plasmid RET.

Fig.2 getting the hybrid protein PSH. And - accumulation of the protein in E. coli BL21(DE3). B - protein purification on Ni-NTA agarose.

Fig.3. Peroxidase activity of the protein PSH. As control is taken recombinant peroxiredoxin 6 person (Prx6).

Fig.4. Superoxidedismutase activity of the protein PSH. As a control used dismutase.

Fig.5. Localization of endogenous peroxiredoxin Prx6 in the heart. Magnification × 1000. Primary antibodies - polyclonal rabbit antibodies to peroxiredoxin 6; secondary antibodies - antibodies ass to rabbit immunoglobulins, conjugated with alkaline phosphatase (USBiological, USA).

Fig.6. Localization of exogenous protein PSH during reperfusion of the heart in the presence of hydrogen peroxide.

Description

To achieve these objectives and to obtain an effective multifunctional chimeric protein is capable of neutralizing a wide range of reactive oxygen species (bifunctional protein with a combined superoxiddismutase and peroxidase activities), you must make a choice between different types of superoxiddismutase, and various classes and subclasses of peroxidases.

As the first component of the enzyme of the antioxidant would be the chosen protein peroxiredoxin (Prx6), with a broad substrate specificity with respect to hydroperoxides. Peroxiredoxin (Prx1-Prx6) are widely represented in the living world and into the family Veselinovic peroxidases, engaged in the destruction of peroxides both organic and inorganic nature. For catalysis Prx use conservative cysteine residue (Cys) in the active site and does not contain any redox cofactors (heme, flavins or metal ions). The most outstanding representative of the class of peroxiredoxins is peroxiredoxin 6 (Prx6), containing only one active cysteine residue (Cys47). Protein Prx6 found in all mammalian cells, but the greatest number are detected in the cytoplasm of epithelial cells: lungs, respiratory tract, gastrointestinal tract and oral cavity, as well as in cells of the liver and pancreas. Prx6 able to recover a wide range of peroxides, including H2About2small organic hydroperoxides, such as tert-butyl hydroperoxide and the hydroperoxide cumene, hydroperoxides of fatty acids and hydroperoxides of phospholipids. This Prx6 able to recover as free molecules of hydroperoxides of fatty acids and the composition of the phospholipids. Peroxidase activity Prx6 person is from 1.8 to 5 µmol/min/mg protein. Unlike about what the rest of peroxiredoxins, Prx6 in addition to peroxidase activity in the acidic environment of the active CA2+independent phospholipase A2 (iPLA2), which may play an important role in the metabolism of phospholipids epithelial cells of the lung [Fisher, A. C., C. Dodia, Feinstein, S. I., But Y. S. Altered lung phospholipid metabolism in mice with targeted deletion oflysosomal-type phospholipase A2. J. Lipid Res. Vol.46, pp.1248-1256 (2005)].

For the task of neutralizing all major types of reactive oxygen species belonging to the group consisting of superoxidation, inorganic and organic hydroperoxides and peroxynitrite, as the second component of the bifunctional chimeric protein was selected as one of the representatives of superoxiddismutase - mn containing superoxide dismutase from E. coli (SOD2) [Roberts, Hirst R. Identification and characterization of a superoxide dismutase and catalase from Mycobacterium ulcerans. J. Med. Environ. Vol.45, pp.383-387. (1996)].

Superoxidedismutase activity (F) was ~9 units/mg protein, which almost corresponds to the expected size of ~17 units/mg protein Mn-SOD from E. coli [C. Roberts and R. Hirst, 1996], where one unit corresponds to the amount of protein required for the manifestation of 50% dismutation of superoxidation.

The obviousness of technical solutions to obtain bifunctional protein PSH presented in this invention is that the protein peroxiredoxin has a complex tertiary structure and the pairing of the two proteins SOD and Prx6 could lead crescom reduction of antioxidant activity of the new complex.

Another obvious characteristic of the selected structure of the bifunctional protein PSH is its stability and preservation as peroxidase and superoxiddismutase activity, for example, when working in the intercellular space.

The next obvious feature of the bifunctional complex PSH is its high efficiency as a component for reperfusion of organs of mammals.

The development of recombinant chimeric bifunctional protein-antioxidant is genetic engineering methods by combining genes peroxiredoxin and superoxide dismutase. In this case, the relationship between proteins is strictly controlled way in the field of irregular parcels and has virtually no effect on the spatial structure of the component structures, which allows to preserve the enzyme activity for each component of the enzyme.

For genetically engineered constructs for the expression of bifunctional protein PSH selected combination of the following enzymes-antioxidants: human peroxiredoxin Prx6 and manganese superoxide dismutase (MnSOD) from E. coli.

In General, the method of obtaining genetically modified bifunctional protein PSH consists of the following stages:

A) produce DNA encoding peroxiredoxin Prx6,

B) allocate DNA to drowsey the superoxide dismutase (MnSOD) from E. coli,

B) choose the type of the plasmid and its genetic design,

D) form a plasmid

E) select the cells and carry out the transformation of cells obtained plasmid,

G) growing the cells on the surface apparitional environment and provide the subsequent growth of cells in liquid medium,

C) release of genetically modified bifunctional protein PSH from the destroyed cells and conduct subsequent protein purification PSH.

In the first step of obtaining a composite design of bifunctional protein PSH produce DNA encoding peroxiredoxin Prx6, and DNA encoding the superoxide dismutase (MnSOD) from E. coli.

Gene peroxiredoxin Prx6 obtained as a PCR product using as template a plasmid containing the cDNA of the gene peroxiredoxin Prx6 [U.S. patent No. 8003345, Fesenko, et al. Antioxidant pharmaceutical compound, method for producing polypeptide and method of cure, publ. 23.08. 2011], and primer Peri (SEQ ID NO:2)

5'-TTTCATATGCCCGGAGGTCTGCTT;

and primer Per Sod 2 (SEQ ID NO:3)

5'-CAGGGATGGCAGGGTATAGCTAGGCTGGGTGTGTAGCGG.

Primer peri (SEQ ID NO:2) contains at the beginning of the sequence of customers recognition of restrictase Nde I (highlighted in underlined fragment) and the beginning of the gene peroxiredoxin Prx6. Thus, primer per1 complementary 5' region of the gene peroxiredoxin 6 persons.

Primer perSod 2 consists of two sequences - end of the gene peroxiredoxin (highlighted underlined fragment) and the beginning of the gene su is peroxydisulfate (MnSOD) E. coli.

The second part of the gene was obtained as a PCR product using as a template DNA of E. coli With and primer perSod I (SEQ ID NO:4)

5'CCGCTACACACCCCAGCCTAGCTATACCCTGCCATCCCTG

and primer sod 2 (SEQ ID NO:5)

5' GCGGCACGTTTTGCGGCGAAAAAACTCGAGTTT.

Primer sod 2 complementary 3' region of the gene of manganese superoxide dismutase from E. coli. In the primer end of the MnSOD gene and restriction site Xho I highlighted underlined fragments.

Mode PCR: the heating of 94°C for 2 min, then 30 cycles - warming 94°C - 30 sec, annealing of primers at 58°C - 30 sec, the synthesis of 68°C for 60 sec. At the end of the PCR - completion 5 min 68°C. the PCR Product was subjected to electrophoresis in 0.8% of agarose, and the band containing the desired fragment was cut from the gel. DNA was isolated and purified using QIAGEN columns (Germany) according to the instructions of the manufacturer.

Purified fragments were mixed (5 ng in 50 μl) and hybridized (annealed) 2 min 95°C followed by 5 min 52°C, and so 5 times. Then the reaction mixture were introduced two primers peri and sod 2 and PCR was performed. Mode PCR: the heating of 94°C for 2 min, then 30 cycles - warming 94°C - 30 sec, annealing of primers at 58°C - 30 sec, the synthesis of 68°C for 60 sec. At the end of the PCR - completion 5 min 68°C. the PCR Product was subjected to electrophoresis in 0.8% of agarose, and the band containing the desired fragment was cut from the gel. DNA was isolated and purified using QIAGEN columns (Germany) according to the instructions of the manufacturer. The floor is obtained DNA fragment was treated with restrictase NdeI and XhoI and again subjected to electrophoresis in 0.8% of agarose. The band of the desired fragment was excised and purified on a column to remove the enzymes. The resulting fragment was Legerova in plasmid rat treated the same restrictases. Thus was obtained a plasmid pET23(PSH), encoding a protein compound PSH molecular weight of 45 KD, amino acid sequence shown in SEQ ID NO:1.

The plasmid contains the promoter of the RNA polymerase of phage T7, the site of initiation of replication (ori) and the terminator of transcription of the ribosomal operon genetic marker - Ar, determining the stability of the transformed plasmid of E. coli cells to ampicillin, a unique recognition sites of restriction endonucleases, as well as a sequence encoding a 6 his-tag residues immediately adjacent to the end of the gene PSH.

Obtained and purified protein PSH applied when conducting reperfusion injury in the isolated heart. This option is the application of protein PSH includes but does not limit other uses for protein PSH.

Protein PSH dissolved in a solution of sodium chloride, preferably isotonic (0.9% NaCl), protein PSH can also be dissolved in a known preservative solutions, such as solutions of Krebs-Henseleit, UW, Art. Thomas II, Collins, Stanford and the like. This solution can be used during all phases of the transplant organ is in, especially the heart, including the following (but not limited to): 1) isolation of an organ from a donor; 2) preservation of authority (storage and transport in hypothermia) and (3) the implantation of an organ recipient (solution for reperfusion).

For solution preparation first prepare 10-fold concentrates of all components (mother solution). To do this in a graduated bottle with a capacity of 1 l pour 700-800 ml of bidistilled water and dissolve in series (to avoid the formation of insoluble precipitation) following chemically pure salt (in grams). As one of the options in table 1 shows the solution for reperfusion injury in the isolated rat heart. The concentration of all protein types in reperfusion fluid - 0.5 mg/ml

Table 1
The solution for reperfusion injury in the isolated rat heart
Saltg/l
NaCl80,0
KCl4,0
MgSO4*7H2O1,0
MgCl-6H2O1,0
Na2 40,6
or Na2HPO4*2H2O0,75
or Ka2PO4*N2O1,5
KH2PO40,6
Glucose10,0
CaCl21,4
or CaCl2·H2O1,85
or CaCl2·6N2Owas 2.76
Phenol red0,2
GasSaturation of the air

To prevent sedimentation of the solution of calcium chloride is prepared separately and added slowly to a solution of the other salts after complete dissolution. Received basic solution lead digitalnirvana water to 1 l), filtered through filter paper and sterilized by passing through a filter of 0.45 and 0.22 microns.

When reperfusion through the aorta isolated rat heart (with forced pumping Hanks solution) in the control of steady heartbeat of the heart with a frequency of 80-100 beats/min During prolonged reperfusion (more hours) heart rate slightly decreases.

Adding on Wednesday Hanks hydrogen peroxide (artificial oxidative stress) there is a rapid (within the first minute) a significant increase in heart rate (up to 125-140 beats/min). Thus, externally, the effect of exogenous injection into the blood stream peroxide is manifested in the increased heart rate. However, after 15-30 min is the cessation of the heartbeat.

When added to the perfusion medium Hanks recombinant peroxiredoxin 6 there has been a slight increase in heart rate (up to 125 beats/min is better myocardial contractility. Washing entered peroxiredoxin 6 stores the frequency of contraction of the isolated heart while maintaining the other parameters. Introduction after washing peroxiredoxin 6 in the Hanks solution of hydrogen peroxide did not cause any effect on the frequency and quality.

When introduced into the environment Hanks bifunctional protein PSH a decrease in heart rate (up to 50 strokes/min). Externally there is better myocardial contractility. The hillshade introduced chimeric bifunctional protein PSH leads to an increase in the frequency of contraction of the isolated heart to 85-90 beats/min while maintaining the other parameters. The introduction of hydrogen peroxide in the Hanks solution after washing the bifunctional protein PSH slightly increases what about the heart rate (up to 100 beats/min), that corresponds to the heart rate in the control experiments.

The full Protocol of the results obtained are shown in table 2.

Table 2
The Protocol of the received data reperfusion
ImpactThe frequency of heart. abbr. (beats/min)Mechanics of the heart
Control60 (initial)
38 (end)
Good
H2O280 (initial)
130 (after H2O2)
Weak
Peroxiredoxin 684 (after washing)
90 (after protein)
55 (after H2O2)
Good
Peroxiredoxin-SOD88 (after washing)
76 (after protein)
55 (after H2O2)
Good

Immunohistochemical studies showed that the normal endogenous peroxiredoxin Prx6 is localized mainly in the blood vessels of the heart (see Fig.5), and on the outer side of the vessel, which primecheck connective tissue and directly to the myocardial cells. The action of hydrogen peroxide leads to some increase in the number of peroxiredoxins in the vessels of the heart, which, apparently, is associated with increased expression of peroxiredoxin in conditions of oxidative stress.

With the introduction of exogenous peroxiredoxins Prx6 and protein PSH during reperfusion of the heart after half an hour after the start of reperfusion proteins are also found mainly in the blood vessels of the heart, and on the outer side of the vessel (see Fig.6), which is adjacent to the connective tissue and directly to the myocardial cells. Obvious fact is that exogenous peroxiredoxin Prx6 and protein PSH adsorbed in the same places, which is predominantly localized and endogenous peroxiredoxin Prx6. This leads to a significant increase in the antioxidant status of blood vessels and adjacent to the vascular tissue of the heart.

Below are examples 1-7, which illustrate the steps of forming plasmids, transformation and cultivation of cells, and the protein purification. These examples include, but are not limited to specific conditions and parameters scope of the invention.

Example 1. The choice of the type plasmid and its genetic structure and formation of plasmids

Thus obtained PCR fragment, which includes genes peroxiredoxin 6 and manganese superoxide dismutase, cloned in expressyou the s vector pET23b points enzyme NdeI and XhoI.

The test structures for the presence of nucleotide substitutions was performed using sequencing in the firm Evrogen (http://evrogen.ru).

The sequencing results were analyzed using Chromas and Fasta (http://www.ebi.ac.uk/Tools/sss/fasta/nucleotide.html).

The analysis of the resulting structures showed that indeed there was obtained a structure in which the genes peroxiredoxin 6 and superoxide dismutase are consistently and not have nucleotide substitutions. The resulting construct was used for transformation expressing the E. coli strain BL21(PSH) and the subsequent developments of the hybrid protein.

Example 2. Cloning of genetically engineered structures in bacterial or yeast genome

2.1. Ligation

40 ng of plasmid DNA were mixed with 40 ng of PCR fragment in a volume of 50 μl. Added 5 μl of ligase buffer and 5 units of ligase. In the control instead of the snippet was just water. Ligation took place at 18°C for 9 hours.

2.2 Transformation

Bacteria BL21(DE3) pre-grown overnight from a single colony. To transform grown fresh culture night culture was diluted in fresh medium 1:100 and grown in the rocking chair for about 3 hours before the appearance of visible turbidity. 8 ml svezhevykrashennyh bacteria are centrifuged and the precipitate washed with 0.1 M calcium chloride. Finally the cells were suspended in 0.4 m is 0.1 M CaCl 2. Operations are conducted strictly in the cold (4°C). Cold mix ligase reaction with 200 μl of bacteria and incubated on ice for 40 min, then transferred to a water thermostat at 41°C for 60 sec, and then cool and add 800 ál of LB medium and incubated on a shaker at 37° for 1 hour. Do the sowing of 10 and 100 µl on Petri dishes with LB medium and adding 100 units/ml of ampicillin and 20 units/ml of chloramphenicol. Cup incubated over night at 37°C.

2.3 Selection of the clones

In the result, we selected 3 of the clone containing an insert of the right size and a compound expressed protein. For these three clones was carried out by sequencing of the insert in order to verify that the cloned composite gene has the correct sequence. Sequencing was performed by Evrogen using capillary sequencing machine. In one case, the sequence of the gene was an error, the other two received an exact match with the sequence represented in GenBank.

One of these clones was selected for further work. Its nucleotide sequence length 1290 p. O. and amino acid sequence length of 435 amino acids in the expressed protein PSH shown in sequence listing SEQ ID NO:1. Molecular weight PSH is approximately 47 kDa (Prx6 - about 25 kDa, Mn-SOD of about 22 kDa). The joining place of the gene peroxiredoxin the ina (ends on GCCT) and SOD gene (starts with ATGAGC) corresponds to positions 672-673 p. on in the sequence listing SEQ ID NO:1.

Example 3. Checking the efficiency of transformation and genetic stability of the producer

To test the genetic stability of night the culture of clone plated on agar with antibiotics and without them and count the number of colonies grown on cups with seed from the same breeding. One of the colonies grown on agar with antibiotics, again seeded in culture medium and again count the number of colonies grown on agar with antibiotics.

For this purpose, 100 μl of the suspension of bacteria from stock grown from a single colony and stored at -75°, were seeded in 50 ml of LB medium. Incubation continued for about 16 hours (overnight) on a rocker at 37°. From the resulting suspension do control the sowing of the Cup with antibiotics (ampicillin 100 units/ml and chloramphenicol 20 units/ml), and the rest of the material introduced in 1.8 l of LB medium with the addition of the same antibiotics in the same concentrations. In the control sowing checks for the presence of plasmids: the same number of colonies on the control and cups with antibiotics indicates the stability of the strain and the presence of plasmids.

All was done and analyzed 8 of such passages. The results are presented in table 3.

Table 3
Genetic stability of the clone PSH1
The number of passageColonies on agar with antibioticsAverageColonies on agar without antibioticsAverage
1221, 208, 230220215, 225, 230223
2189, 195, 200195183, 190, 206193
3301, 306, 290299295, 298, 320304
4250, 265, 220245248, 218, 230232
5350, 330, 342344356, 340, 320339
6178, 184, 196186180, 173, 190182
7370, 352, 360364 365, 342, 375371
8320, 299, 340320325, 296, 345323

As can be seen from table 3, for all passages not found loss of the plasmid that encodes resistance to ampicillin.

The culture of the last passage was tested for the induction of protein synthesis PSH, and confirmed by normal induction of protein expression PSH. Thus, a strain of E. coli for many generations retains cloned gene.

To verify the integrity of the cloned gene in medium-term storage of a producer svejeviratna night culture PSH were stored in a refrigerator at 4°C. Periodically make sowing on agar with ampicillin (100 units/ml) and chloramphenicol (20 units/ml), as well as on agar without antibiotics out of 10(-7) cultivation. The next day, count the number of colonies.

Cultivation of the strain produced in the fermenter for 2 l at 37° C under intensive aeration to achieve a turbidity of about 0.4. Then make IPTG to a final concentration of 1 mm and continue incubation for another 2 hours.

Bacteria are harvested by centrifugation (4000 rpm, 20 min), the precipitate frozen at -20°C.

Example 4. Isolation and purification of protein SPH

The presence of the His-tag in the protein structure PSH simplifies cleaning combinatio protein due to the fact, this domain specifically binds to Ni-NTA-agarose.

Residue biomass of bacteria E. coli resuspending in 25 ml of buffer for application to the column 12 mm Tris-HCl, pH 7.8, 10 mm imidazole) and destroy ultrasound at 4°C. the Cell debris is removed by centrifugation at 14000 rpm for 10 min, adosados filtered through a 0.45 μm filter (Corning, USA) and applied to a column (BioRad, USA) with 10 ml Ni-NTA-agarose (Invitrogen, USA), which was previously equilibrated with buffer for drawing. Incubation of the bacterial lysate with Ni-NTA-agarose carried out for 40 min at +4°C. Then, the column was washed with 100 ml: 12 mm Tris-HCl pH 7.8, 20 mm imidazole. The elution of protein spend 5 ml buffer: 12 mm Tris-HCl, pH 7.8, 250 mm imidazole. Protein is concentrated using a membrane concentrator VIVASCIENCE 30.000 MWCO (Sartorius, Germany) and cialiswhat against 1×PBS (1.7 mm KH2PO4, 5.2 mm Na2HPO4, 150 mm NaCl, pH 7.4). The purity of the protein, as measured by polyacrylamide gel electrophoresis, was about 95%. Usually get about 20-40 mg of purified protein from one fermentation. Electrophoresis of proteins carried out under denaturing conditions (in the presence of SDS) using the standard method of Laemmli [Laemmli, U. K. 1970, Nature, 227, 680-685] equipment Mini Vertical Unit (Amersham, USA). We use 5% of concentrating and 12.5% allowing polyacrylamide gels (SDS page) in Tris-buffer (130 mm Tris-HCl, pH 68, for concentrating and 375 mm Tris-HCl, pH 8.8, for permitting)containing 0.1% SDS, 0.1% PSA 0.08% TEMED. Electrode buffer is a solution: 25 mm Tris, 250 mm glycine and 0.1% SDS. Electrophoresis in concentrating the gel is carried out at current 15 mA (10-15 min), then in permissive gel at 25 mA (40-45 min). Staining of the gel is carried out using Comassie Brilliant Blue R-250 ("Fluka, USA). In Fig.2 shows the data of the hybrid protein electrophoresis PSH. Where a is the accumulation of the protein in E. coli BL21(DE3). B - protein purification on Ni-NTA agarose.

Example 5. Determination of enzyme activities, protein SPH

5.1. Peroxidase activity of the protein SPH against hydrogen peroxide and tert-butyl hydroperoxide

Peroxidase activity determined according to [Kang S. W., Baines I. S., Rhee, S. G. (1998). Characterization of a mammalian peroxiredoxin that contains one conserved cysteine. // J. Biol. Chem. Vol.273, pp.6303-6311] with minor modifications, using as substrates the hydroperoxide hydrogen (inorganic hydroperoxide and tert-butyl hydroperoxide (organic hydroperoxide). As a negative control use peroxiredoxin 6 man with the replacement of the cysteine residue in the active center of the series (Cys47Ser). The reaction mixture (150 μl) contains 7 mm phosphate buffer, pH 7.3, 150 mm NaCl, 2 mm DTT, 100 μm hydrogen peroxide or 100 μm tert-butyl hydroperoxide (the state) and the enzyme at various concentrations. The reaction is carried out for 10 min at 37°C and East Jerusalem. nalivajut, adding 50 ál of 0.6 M HCl. Then add 100 ál of 10 mm Fe(NH4)2(SO4)2and 50 μl of 2.5 M KSCN, which leads to the formation of complex compounds of iron red. The peroxide concentration is proportional to the intensity of the color, measured at a wavelength of 492 nm on the device Multiscan. An example of such a definition is shown in Fig.3 Peroxidase activity PSH against hydrogen peroxide 100 nmol/min/mg, which is approximately twice lower than for pure peroxiredoxin.

5.2. Superoxidedismutase activity

Superoxidedismutase activity determined using a commercial kit Superoxide Dismutase Assay Kit (Calbiochem, USA), in accordance with the manufacturer's instructions.

Superoxidedismutase activity PSH was about 9 units/mg protein, see Fig.4, which is almost equal to the expected value of about 17 units/mg protein Mn-SOD from E. coli [C. Roberts and R. Hirst, 1996], where one unit corresponds to the amount of protein required for the manifestation of 50% dismutation of superoxidation.

Taking into account that the molecular weight PSH approximately 47 kDa (Prx6 about 25 kDa, Mn-SOD about 22 kDa), 1 mg PSH approximately 2 times smaller molecules Prx6 and Mn-SOD than 1 mg protein Prx6 or Mn-SOD, therefore, when calculating the activity of 1 mg PSH is expected to double reduction activities. Thus, we can conclude that both peroxidase and superoxiddismutase AK is Yunost has been completely preserved in the protein PSH.

Example 6. The cultivation conditions when receiving preparative quantities of recombinant protein PSH

When developing methods for culturing E. coli with downregulation of protein PSH, emphasis was given to obtaining conditions under which there is the maximum expression of SPH protein with minimal expression of its own proteins in E. coli.

Strains of E. coli:_BL21(DE3)[pRIL] Codon Plus. Genotype: F-dcm ompT hsdS (rB- mB-) gal λ (DE3) [pRIL CamR] ("Stratagen, USA). This strain is a derivative of BL21 containing plasmid pRIL, which determines resistance to chloramphenicol and contains 3 genes tRNA that recognizes the codons of arginine (R), isoleucine (I) and leucine (L), characteristic of eukaryotes. Strain lithogenes on λDE3, which contains the gene for T7 RNA polymerase under control of the lacUV5 promoter (inducible - IPTG, galactose; repressor - glucose). The transformation efficiency of the competent cells (obtained CA2+method) to 1×107the transformants per 1 μg of plasmid DNA pUC19. This strain is usually used to improve the synthesis of eukaryotic proteins in E. coli cells. Cultivation in liquid medium LB+chloramphenicol (35 μg/ml), on a solid nutrient medium LB+agar+chloramphenicol (35 μg/ml). For transformation of bacteria BL21(DE3) pre-grown overnight from a single colony. To transform grown fresh culture night culture was diluted in with the hedgehogs environment 1:100 and grown in the rocking chair for about 3 hours before the appearance of visible turbidity. 8 ml svezhevykrashennyh bacteria are centrifuged and the precipitate washed with 0.1 M calcium chloride. Finally the cells were suspended in 0.4 ml of 0.1 M CaCl2. Operations are conducted strictly in the cold (4°C). Cold mix ligase reaction with 200 μl of bacteria and incubated on ice for 40 min, then transferred to a water thermostat at 41°C for 60 sec, and then cooled, add 800 ál of LB medium and incubated on a shaker at 37° for 1 hour. After screening of transformants determine gene expression PSH. For each clone inoculated into a test tube Falkone 50 ml in a volume of 10 ml with the addition of antibiotics and grown at rocking up to an optical density of 0.5, and then make IPTG to a concentration of 1 mm and continue growing for another 3 hours. After that, select 1 ml, centrifuged at 10,000 rpm for 5 minutes the Precipitate resuspended in 50 μl of buffer and carry out electrophoresis. In the presence of IPTG is the induction of gene expression under T7 promoter and the bacterial protein produced PSH. Typically, the protein yield is about 30% of the total soluble protein that is easily detected by electrophoresis. In the result, we selected 3 of the clone containing an insert of the correct size, and expressed the gene. For these three clones was carried out by sequencing the gene in order to verify that the cloned gene is polyhistidine and the right pic is egovernance. Sequencing was performed by Evrogen using capillary sequencing machine. In one case, the sequence of the gene was an error, the other two received an exact match with the sequence represented in GenBank. One of these clones was selected for further work. In the future, this clone was named E. coli BL21(PSH).

Example 7. The use of bifunctional protein PSH during reperfusion of the heart

Clip blocking the flow of the perfusion fluid into the cannula, open fully, than increase perfuziruemah solution into the cannula until 12-15 ml/min. Then cut off the light. In the perfusion and cardioplegic solution from the aorta flows into the coronary artery that nourishes the heart and goes through the veins of the heart into the right atrium. When the contraction of the right atrium and right ventricle solution flows through the pulmonary trunk, upper and lower hollow veins, cleansing the heart.

The introduction of the hydrogen peroxide solution at a concentration of 200 μm in the bloodstream dramatically changes the rhythm of the heartbeat. Dramatically increases the heart rate (two times compared to control) after the introduction of the peroxide. This effect develops rapidly during the first minute. Further reperfusion leads to the cessation of heartbeat after 30 minutes. Thus, there are objective parameters that allow control shall be a condition of the heart muscle.

Introduction to reperfusion solution of bifunctional protein PSH in a concentration of 0.1-0.5 mg/ml and subsequent reperfusion and removal is not bound peroxidase protein of perfusion environment significantly improves the condition of the heart muscle. Subsequent introduction of reperfusion peroxide solution is not observed its effect on the heart activity. It should be noted that in these experiments the protein PSH was not present in the bloodstream, since before adding hydrogen peroxide in the reperfusion solution free protein PSH was completely removed by washing from the heart. Thus, the effect of protein PSH due to the amount of protein PSH, who was adsorbiroval in the heart during reperfusion.

Direct measurement of the content of all investigated peroxiredoxins adsorbed in the tissues of the heart during reperfusion, showed that the heart is adsorbed by the relatively small number of peroxiredoxins. The essential point is the fact that most effectively peroxiredoxin by reperfusion are sorbed in the atrium, but not in the ventricle. Apparently, this explains the strong and rapid effect of protection of the heart from the action of hydrogen peroxide (see below).

As mentioned above, when reperfusion occurs sorption protein PSH in different parts of the heart. In this case, you can rely on a significant higher is their antioxidant status in tissues of the heart. In the control when introduced into the perfusion solution of hydrogen peroxide is observed a sharp increase of lipid peroxidation in the tissues of the heart (5-8 times). Pre-reperfusion protein PSH leads to a complete blocking of additional lipid peroxidation induced by hydrogen peroxide. So, really recombinant protein PSH provides a prolonged effect of increasing the antioxidant status of the body, at least in the case of the heart.

Industrial applicability

Protein PSH, expressed in E. coli, has as peroxidase and superoxiddismutase activities, similar molar activities of human peroxiredoxin 6 and yeast superoxide dismutase (100 nmol/min/mg of tert-butyl hydroperoxide and ~9 units/mg superoxidation).

Check the transformation efficiency of E. coli showed that induction of expression in E. coli recombinant protein PSH using IPTG this protein is a major and approximately 30% of all soluble proteins.

Downregulation of protein PSH is a water-soluble (and not contained in the inclusion bodies of E. coli), which can significantly simplify the procedure selection using affinity chromatography.

Thus, the producer bifunctional chimeric protein PSH with somese the governmental peroxidase and superoxiddismutase activities on the basis of E. coli. himself highly purified protein PSH.

The obtained protein PSH promotes efficient recovery of cardiac activity after prolonged ischemia, reduction of metabolic disorders and contractural myocardial injury.

1. Chimeric nucleic acid encoding a hybrid protein PSH, including human peroxiredoxin Prx 6 and the manganese superoxide dismutase MnSOD, possessing antioxidant activity of superoxide dismutase and peroxidase, primary structure which describes the amino acid sequence SEQ ID NO:1.

2. Chimeric nucleic acid under item 1, which is obtained by cloning of individual gene fragments Prx 6 and MnSOD in a PCR reaction using the synthetic oligonucleotides SEQ ID NO:2 - SEQ ID NO:5.

3. The recombinant expression vector pET23(PSH)containing the chimeric nucleic acid encoding a protein PSH with the amino acid sequence SEQ ID NO:1, operatively integrated into a plasmid pET22b(+) restriction sites Nde I and Xho I, C-terminal localization of polyhistidine.

4. A hybrid protein PSH for reperfusion of the heart, the primary structure of which is described amino acid sequence of SEQ ID NO:1.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to recombinant obtaining G-CSF, and can be used for production of G-CSF in cells of E.coli. For effective production of protein in cells of E.coli G-CSF-coding DNA sequence is optimised. On the basis of obtained optimised DNA sequence plasmid pAS017, also including NdeI/BamHI-fragment of DNA of pETM-50 vector and having physical map, presented on the drawing, is constructed.

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2 cl, 1 dwg, 1 tbl, 3 ex

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SUBSTANCE: invention refers to biotechnology, particularly to genetically engineered production of human proteins, and may be used for preparing human epidermal growth factor (hEGF) in bacterial cells in the form of glutathione-3-transferase fusion protein. What is constructed is the recombinant DNA coding GST-hEGF fusion protein which consists of an amino acid sequence of glutathione-S-transferase and an amino acid sequence of human epidermal growth factor divided by a cleavage site by enterokinase, and characterised by the nucleotide sequence SEQ ID NO:1. The KpnI/XhoI fragment of the vector pET41 and the above recombinant DNA are used to create the recombinant plasmid pAS007 for expression of GST-hEGF fusion protein in E.coli cells.

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SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

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FIELD: medicine.

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FIELD: biotechnologies.

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SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, particularly toxicology and radiology, to drug preparations based on antioxidant proteins and methods of using them. The pharmaceutical composition for treating toxic conditions wherein the therapeutic effect is ensured by the action of antioxidant, antimicrobial, antitoxic human lacroferrin protein on the human body contains non-replicating nanoparticles of human adenovirus serotype 5 genome with inserted human lactoferrin expressing human lactoferrin in the therapeutically effective amount in the body, and an expression buffer with the particle content not less than 2.33×1011 of physical particles per ml of the expressing buffer. The method of therapy involves administering the composition in the therapeutically effective dose of 7×1011 of physical particles to 7×1013 of physical particles per ml of the expressing buffer per an individual; the composition is administered intravenously.

EFFECT: invention provides the stable therapeutic effect after the single administration of the composition.

17 cl, 14 ex, 4 dwg

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

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9 cl, 5 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns a method for the prediction of a human body length within the Russian population. The presented method is based on the DNA analysis, wherein the PCR method and specific primers are used to examine amelogenin (AML) gene sites with using gene loci of a secretory growth hormone receptor (GSHR), a ligand-dependent corepressor-like nuclear receptor (LCORL) and a binding protein of cycline-dependent kinase (CABLES1) in male DNA samples and genes of a hedgehog-interacting protein (HHIP) and a nuclear protein with zinc fingers (JAZF1) in female DNA samples. The body length is shown by inserting the derived values into a specific formula that is followed by mathematical processing of the analysis results.

EFFECT: solution can be used in genome dactylography, medical genetics, molecular-biological studies within the prediction of phenotypic signs by considering genetic data in the population.

7 dwg, 1 ex

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