Method of obtaining recombinant mutant cytochrome c

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

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The invention relates to the field of biochemistry, molecular biology and biotechnology. Can be used for measuring the rate of generation of superoxide in membrane preparations (microsomes, mitochondria)with cytochrome C reductase and cytochrome C oxidase activity, as well as in medical biochemistry to study the role of superoxide in the pathogenesis of neurodegenerative diseases.

Cytochrome C is a small protein with covalently attached zhelezovanadievoj group. In eukaryotes cytochrome C is localized on the surface of the inner mitochondrial membrane facing the intermembrane space. Its main function is to participate in the transfer of electrons from original: cytochrome C oxidoreductase (complex III) cytochrome C oxidase (complex IV) of the respiratory chain. Furthermore, known for its ability to oxidize superoxide. Mutant variants of cytochrome C, deprived of respiratory function, but retained the ability to oxidizeare a potential basis for the creation of test systems for the determination of superoxide.

A method of obtaining cytochrome C horse from the heart tissue (Dickerson R.E., M.L. Kopka, Borders C.L., J. varnum poor, Weinzierl J.E., Margoliash E.A centrosymmetric projection at 4 Å of horse heart oxidized cytochrome C // J. Mol. Biol. 1967. Vol.29, pp.77-80).

A method of obtaining recombinant cytochrome C horse (Long is A, Latypov Russia, having got, Abdullayev SH, colon, C., Roder H., Kirpichnikov M.P. Expression of mutant genes of cytochrome C horse in Escherichia coli // Bioorganic chemistry, 1998. 24, No. 10, s-759).

However, cytochrome C wild-type, added to the mitochondrial preparations (in particular, to submitochondrial particles), is restored to a greater degree components of the respiratory chain than superoxide, and, therefore, cannot serve as a reliable agent for the quantitative determination of the rate of generation of superoxide.

A method of obtaining partially acetylated cytochrome C (ACC), (Azzi A., Montecucco C., Richter .The use of acetylated ferricytochrome c for the detection of superoxide radicals produced in biological membranes // Biochem. Biophys. Res. Commun. 1975. Vol.22, pp.597-603). Partially acetylated cytochrome C is produced by a chemical reaction between cytochrome C and acetic anhydride. ACC has a reduced ability to redox reactions with components of the respiratory chain of mitochondria and microsomes. ACC is restored mitochondrial or microsomal reductase and oxidized by cytochrome oxidase with lower rates compared to cytochrome C wild type, the ability of ACC to restore the superoxide is stored.

The disadvantage of this method is that when the acetylation of cytochrome C with acetic anhydride, a sufficiently heterogeneous mixture of molecules is of akroma with different number of modified lysine residues. Reactivity of cytochrome C in relation to natural reductants and oxidants depends on the degree of acetylation. When the acetylation reduces the overall positive charge of cytochrome C, which leads to reduced affinity to superoxide. The nonspecific interaction of the ACC with components of the respiratory chain is reduced by the addition of phosphate or potassium chloride in relatively high concentrations (up to 100 mm), however, high concentrations To+cause partial separation of the respiratory chain, which leads to a lower real rate of generation of superoxide. These deficiencies ACC lead to low accuracy rate of generation of superoxide when using ACC.

The invention solves the problem of obtaining a recombinant mutant of cytochrome C horse with a low capacity for redox reactions with components of the respiratory chain.

The task is solved by the method of producing a recombinant mutant of cytochrome C horse, including the introduction of mutations K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K using the method of site-directed mutagenesis based on cyclic polymerase reactions, gene cytochrome C horse, being part of the plasmid DNA pBPCYCS/3, followed by transformation of Escherichia coli strain JM-109 received the Oh recombinant plasmid DNA, the expression and secretion of the target protein using cation exchange and adsorption chromatography.

A method of obtaining a recombinant mutant of cytochrome C horse-based design of mutant gene cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K with amino acid substitutions at the site of interaction with redox partners of the respiratory chain: original: cytochrome C oxidoreductase (complex III) and cytochrome C oxidase (complex IV). Due to the replacement polozhiteljneje lysine residues (K)surrounding the heme cavity of cytochrome C wild-type, negative balances glutamic acid (E) reduced ability to redox reactions with complex III and complex IV. Approach to directional changes of surface charges of cytochrome C in order to "move" of the binding site from the heme cavity to the periphery. To conserve the total positive charge of the protein outside of the site of interaction are introduced back replacement (E→K).

Plasmid DNA pBPCYCS/3 is obtained by embedding on sites Xho I and Wat I gene cytochrome C horse (CYCS), obtained by PCR with plasmid Rav, plasmid pBP(Xho 1/Watn I)/CYC3 (Dolgikh D.A., Latypov Russia, having got, Abdullayev SH, colon, C., Roder H., Kirpichnikov M.P. Expression of mutant genes of cytochrome C horse Escherichia coli // Bioorganic chemistry, 1998. 24, No. 10, s-759).

Plasmid DNA pBPCYCS/3 contains a mutant gene cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 13) or K8E/E62K/E69K/K72E/K86E/K87E (SEQ ID NO 14), or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 15), built between restriction sites Xho I and Wat I encoding the synthesis of mutant recombinant protein cytochrome C K27E/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 13) or K8E/E62K/E69K/K72E/K86E/K87E (SEQ ID NO 14) or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 15), lac promoters and trc before gene cytochrome C bla gene for synthesis of β-lactamase (resistance to ampicillin), gene yeast heme-ligase (CYC3)that encodes an enzyme involved ligation of recombinant cytochrome C with gem bacterial cells.

Plasmid DNA pBPCYCS/3 containing the mutant gene cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, transform cells of Escherichia coli strain JM-109. Spend the expression of target protein in bacterial cells, its isolation and purification using chromatographica methods (ion-exchange chromatography on a MonoS and adsorption chromatography on hydroxyapatite).

the obtained mutant cytochrome C horse carry out oxidation-reduction reactions with complex III (succinate: cytochrome C reductase) and complex IV (cytochrome C oxidase) of the respiratory chain of mitochondria. Succinate: cytochrome C reductase activity of mitochondria in the presence of ACC with the hat 8% of the activity in the presence of cytochrome C horse wild type. Succinate: cytochrome C reductase activity of mitochondria in the presence of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K is 3% of the activity in the presence of cytochrome C horse wild type (figure 1). Cytochrome C oxidase activity of mitochondria in the presence of ACC is 3% of the activity in the presence of cytochrome C horse wild type. Cytochrome C oxidase activity of mitochondria in the presence of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K not detected (figure 2).

The technical result of the claimed method is that the obtained mutant cytochrome C horse has the ability to redox reactions with components of the respiratory chain lower compared to the ACC and may be used as a test system for measuring the rate of generation of superoxide in membrane preparations (microsomes, mitochondria)with cytochrome C reductase and cytochrome C oxidase activity.

The invention is illustrated figure 1-3:

Figure 1. The dependence of succinate: cytochrome C reductase activity of rat liver mitochondria, specifically depleted by cytochrome C, from the concentration of added protein:

■ - cytochrome C horse wild type, ○ - ACC ◊ - mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, Δ - m is tantoco cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E, * - mutant cytochrome C horse K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K.

Figure 2. The dependence of cytochrome C oxidase activity of rat liver mitochondria, specifically depleted by cytochrome C, from the concentration of added protein:

■ - cytochrome C horse wild type, ○ - ACC ◊ - mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, Δ - mutant cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E, * - mutant cytochrome C horse K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K.

Figure 3. Absorption spectrum of cytochrome C horse wild type and mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K(B).

The invention is illustrated by the following examples:

Example 1. Construction of the mutant gene cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K

For the introduction of mutations K27E/E69K/K72E/K86E/K87E/E90K in the gene of horse cytochrome C in the composition expression plasmid vector pBPCYCS/3 using the method of site-directed mutagenesis based on cyclic polymerase reaction. Introduction of these mutations carried out in four stages.

At the first stage for the introduction of mutations K86E/K87E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3, mutagenity mixture in a volume of 50 µl containing: 5 μl of buffer with MgSO4for Pfu DNA polymerase (Fermentas, Lithuania), 15 ng matrix (plasmid) DNA, 125 ng direct (SEQ ID NO 1) and reverse (SEQ ID NO 2) oligonucleotide primers, 2 mm of each of the four deoxynucleotides and 2.5 units act. thermostable Pfu DNA polymerase (Fermentas, Lithuania). On top of the layer of the reaction mixture of mineral oil in the amount of 30 μl. Spend 20 cycles of amplification reaction according to the following scheme: melting matrix DNA at 95°C (45 s), annealing of primers at 55°C (60 s) and the polynucleotide chain elongation at 74°C (10 min). The accumulation of mutant DNA control using electrophoretic analysis in 1%agarose gel. For splitting the original methylated plasmid DNA 19 ál mutageneses mixture is treated with 2.5 units of the act. restrictase Dpn I buffer (Promega, USA) for 1 h at 37°C. this was followed by a transformation mutageneses mixture of cells of the strain E. coli XL I-Blue, the production of cell biomass and allocation from her plasmid DNA pBPCYCS/3-K86E/K87E. Determination of the nucleotide sequence of the mutant plasmid DNA pBPCYCS/3-K86E/K87E to establish the fact that the introduction of mutations performed on an automated sequencing machine (ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, USA).

In the second phase, carried out the introduction of mutations E90K in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K86E/K87E according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 3 and SEQ ID NO 4.

The third phase will cover the introduction of mutations K27E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K86E/K87E/E90K according to the scheme described above. Using oligonucleotide p is aimery SEQ ID NO 5 and SEQ ID NO 6.

In the fourth stage, carry out the introduction of mutations E69K/K72E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K27E/K86E/K87E/E90K according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 7 and SEQ ID NO 8.

The result is a recombinant plasmid DNA pBPCYCS/3-K27E/E69K/K72E/K86E/K87E/E90K, containing the gene for cytochrome C K27E/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 13)encoding the mutant cytochrome C K27E/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 13).

Example 2. Construction of the mutant gene cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E

For the introduction of mutations K8E/E62K/E69K/K72E/K86E/K87E in the gene of horse cytochrome C in the composition expression plasmid vector pBPCYCS/3 using the method of site-directed mutagenesis based on cyclic polymerase reaction. Introduction of these mutations carried out in four stages.

At the first stage for the introduction of mutations K86E/K87E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3, mutagenity mixture in a volume of 50 µl containing: 5 μl of buffer with MgSO4for Pfu DNA polymerase (Fermentas, Lithuania), 15 ng matrix (plasmid) DNA, 125 ng direct (SEQ ID NO 1) and reverse (SEQ ID NO 2) oligonucleotide primers, 2 mm each of the four deoxynucleotides and 2.5 units of the act. thermostable Pfu DNA polymerase (Fermentas, Lithuania). On top of the layer of the reaction mixture of mineral oil in the amount of 30 μl. Spend 20 is of eklow amplification reaction according to the following scheme: melting matrix DNA at 95°C (45 s), annealing of primers at 55°C (60 s) and the polynucleotide chain elongation at 74°C (10 min). The accumulation of mutant DNA control using electrophoretic analysis in 1%agarose gel. For splitting the original methylated plasmid DNA 19 ál mutageneses mixture is treated with 2.5 units of the act. restrictase Dpn I buffer (Promega, USA) for 1 h at 37°C. this was followed by a transformation mutageneses mixture of cells of the strain E. coli XL I-Blue, the production of cell biomass and allocation from her plasmid DNA pBPCYCS/3-K86E/K87E. Determination of the nucleotide sequence of the mutant plasmid DNA pBPCYCS/3-K86E/K87E to establish the fact that the introduction of mutations performed on an automated sequencing machine (ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, USA).

The second phase will cover the introduction of mutations E69K/K72E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K86E/K87E, according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 7 and SEQ ID NO 8.

The third phase will cover the introduction of mutations K8E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-E69K/K72E/K86E/K87E, according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 9 and SEQ ID NO 10.

In the fourth stage conduct introduction mutations E62K in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K8E/E69K/K72E/K86E/K87E according to the scheme described above. Use oligonucleotid the specific primers SEQ ID NO 11 and SEQ ID NO 12. The result is a recombinant plasmid DNA pBPCYCS/3 - K8E/E62K/E69K/K72E/K86E/K87E, containing the gene for cytochrome C K8E/E62K/E69K/K72E/K86E/K87E (SEQ ID NO 14)encoding the mutant cytochrome C K8E/E62K/E69K/K72E/K86E/K87E (SEQ ID NO 14).

Example 3. Construction of the mutant gene cytochrome C K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K

For the introduction of mutations K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K in the gene of horse cytochrome C in the composition expression plasmid vector pBPCYCS/3 use site-directed mutagenesis based on cyclic polymerase reaction. Introduction of these mutations is carried out in six steps.

At the first stage for the introduction of mutations K86E/K87E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3, mutagenity mixture in a volume of 50 µl containing: 5 μl of buffer with MgSO4for Pfu DNA polymerase (Fermentas, Lithuania), 15 ng matrix (plasmid) DNA, 125 ng direct (SEQ ID NO 1) and reverse (SEQ ID NO 2) oligonucleotide primers, 2 mm each of the four deoxynucleotides and 2.5 units of the act. thermostable Pfu DNA polymerase (Fermentas, Lithuania). On top of the layer of the reaction mixture of mineral oil in the amount of 30 μl. Spend 20 cycles of amplification reaction according to the following scheme: melting matrix DNA at 95°C (45 s), annealing of primers at 55°C (60 s) and the polynucleotide chain elongation at 74°C (10 min). The accumulation of mutant DNA control using the electric is operationsmore analysis in 1%agarose gel. For splitting the original methylated plasmid DNA 19 ál mutageneses mixture is treated with 2.5 units of the act. restrictase Dpn I buffer (Promega, USA) for 1 h at 37°C. this was followed by a transformation mutageneses mixture of cells of E. coli strain XL I-Blue, the production of cell biomass and allocation from her plasmid DNA pBPCYCS/3-K86E/K87E. Determination of the nucleotide sequence of the mutant plasmid DNA pBPCYCS/3-K86E/K87E to establish the fact that the introduction of mutations performed on an automated sequencing machine (ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, USA).

In the second phase, carried out the introduction of mutations E90K in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3-K86E/K87E according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 3 and SEQ ID NO 4.

The third phase will cover the introduction of mutations K27E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3 - K86E/K87E/E90K according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 5 and SEQ ID NO 6.

In the fourth stage, carry out the introduction of mutations E69K/K72E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3 - K27E/K86E/K87E/E90K according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 7 and SEQ ID NO 8.

The fifth phase will cover the introduction of mutations K8E in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3 - K27E/E69K/K72E/K86E/K87E/E90K, according to the scheme described the th above. Using the oligonucleotide primers SEQ ID NO 9 and SEQ ID NO 10.

At the sixth stage, carry out the introduction of mutations E62K in gene equine cytochrome C, being part of the plasmid DNA pBPCYCS/3 - K8E/K27E/E69K/K72E/K86E/K87E/E90K according to the scheme described above. Using the oligonucleotide primers SEQ ID NO 11 and SEQ ID NO 12. The result is a recombinant plasmid DNA pBPCYCS/3 - K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, containing the gene for cytochrome C K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 15), encoding a mutant cytochrome C K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K (SEQ ID NO 15).

Example 4. Production and separation of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K from biomass cells of E. coli strain JM-109 containing recombinant plasmid DNA pBPCYCS/3

The expression of the mutant gene cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, being part of the recombinant plasmid DNA pBPCYCS/3-K27E/E69K/K72E/K86E/K87E/E90K carried out in the strain E. coli JM109 (Bortolotti C.A., M. Borsari, M. Sola, Chertkova R., Dolgikh D., Kotlyar A. and P. Facci Orientation-dependent kinetics of heterogeneous electron transfer for cytochrome with an immobilized on gold: electrochemical determination and theoretical prediction // J. Phys. Chem. C. 2007. Vol.111, pp.12100-12105). Cells transformed by plasmid DNA containing the mutant gene are grown in a liquid nutrient medium SB with ampicillin (final concentration 200 μg/ml) at 37°C With vigorous stirring (250 rpm) for 24 hours

At the end of the gene expression of the mutant is on cytochrome C cells of E.coli precipitated by centrifugation at 4000 g and 4°C for 20 minutes The supernatant is carefully removed, and the residue resuspended in 35 ml of buffer And MonoS (25 mm Na2HPO4, 25 mm NaH2PO4, 1 mm NaN3, pH 6.0) and stored at -20°C.

Homogenization of cells is performed by the burst pressure 2000 psi at the "French Press" (breakers Instruments, Inc., USA). Cell debris is then precipitated by centrifugation at 100000 g for 20 minutes

Isolation and purification of the target protein from the resulting supernatant is performed using liquid chromatography system "BioLogic HR System (BIO-RAD, USA). Cell extract is applied on a cation exchange column Mono S HR 10/10 (BIO-RAD, USA), equilibrated with buffer A. the Mutant cytochrome elute with a linear gradient of buffer for MonoS (25 mm Na2HPO4, 25 mm NaH2PO4, 1 M NaCl, 1 mm NaN3, pH 6.0) at a rate of 3 ml/min

Fractions obtained after purification on Mono S, analyzed spectrophotometrically and by electrophoresis in SDS-page, dialist against buffer And adsorption chromatography (10 mm Na2HPO4, 10 mm NaH2PO4, 1 mm NaN3, pH 7.0) and applied to a column CHT-I (BIO-RAD, USA). Mutant cytochrome elute with a linear gradient of buffer for adsorption chromatography (500 mm Na2HPO4, 500 mm NaH2PO4, 1 mm NaN3, pH 7.0) at a rate of 2 ml/min

The degree of purification and concentration of mutant cytochrome C obtained in fra the operations determined by spectrophotometric and electrophoretic analysis in SDS-page, the most pure fractions are combined oxidized with potassium ferricyanide added in equimolar concentrations, double-cialiswhat against 10 mm ammonium carbonate buffer, pH 7.9 and lyophilized on installing ALPHA 1-5.

The expression of mutant genes of cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E and K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, being part of the recombinant plasmid DNA pBPCYCS/3 - K27E/E69K/K72E/K86E/K87E/E90K and pBPCYCS/3 - K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, respectively, and purification of mutant proteins cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E and K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K carried out according to the scheme described above.

Receive 20 mg of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, 10 mg of mutant cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E and 10 mg of mutant cytochrome C horse K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K with 1 l of cell culture.

To verify the spectral characteristics of the obtained mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K remove the absorption spectrum on the spectrophotometer Saga 50 Bio (Varian, USA) in a medium containing 0.25 M sucrose, 50 mm Tris-HCl (pH 8.0) and 0.2 mm EDTA. For the oxidation of cytochrome C horse wild type (Sigma) or mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K on Wednesday dimension dobavlyaut 20 μm potassium ferricyanide (figure 3, line 1). For cytochrome C reduction horses wild-type or mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K in a spectrophotometric cuvette add a few Crist is low of dithionite sodium (3, line 2). These substitutions do not affect the absorption spectrum of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K. Changing the molar absorption recovery cytochrome C horse wild-type or mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K the maximum at 550 nm is ε550=20000 M-1cm-1(figure 3). The spectral characteristics of the mutant cytochrome C horse K8E/E62K/E69K/K72E/K86E/K87E and mutant cytochrome C K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K identical spectral characteristics of cytochrome C horse wild type and mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K.

Example 5. Testing mutant cytochromes with horses in rat liver mitochondria, specifically depleted by cytochrome C.

5.1. Getting preparation of rat liver mitochondria

Rat weighing 250 g decapitate, liver extract from the opened abdominal cavity. Isolated liver immersed in 30 ml of glacial environment selection, containing 0.25 M sucrose, 1 mm EDTA, pH 7.4. After three times wash cold fabric is placed on a Petri dish placed on ice, and quickly shredded with scissors. Crushed fabric again placed in a fresh environment selection and thoroughly washed. After settling pieces of fabric, the liquid is carefully poured and described operation is repeated twice more.

Washed out so the fabric is transferred to a homogenizer, add 40 ml of medium in the division and homogenized for 40 C. To the resulting homogenate add 40 ml of medium selection is stirred with a slowly rotating pestle and pour the homogenate into two centrifuge Cup. The homogenate was centrifuged at 600 g and 0-2°C for 10 min to remove debris cells and nuclear fractions.

The supernatant is carefully poured and stored on ice, and precipitation are combined and again homogenized for 20 s in 20 ml of medium selection. The homogenate was centrifuged at 600 g for 10 min, and the supernatant combined with previously obtained.

For the deposition of mitochondrial supernatant centrifuged at 14000 g for 10 minutes Obtained precipitates are combined and thoroughly suspended in a small volume of medium allocation (about 0.5 ml). Small portions with gentle shaking add 40 ml of medium selection and precipitated mitochondria (14000 g, 10 min). The precipitate is suspended in 0.25 M sucrose containing no EDTA, and again centrifuged (14000 g, 10 min). The supernatant is poured, and the precipitate mitochondrial carefully layer 0.3 ml of 0.25 M sucrose. Light shaking wash away the layer of loose sediment. This operation is repeated twice more, obtained a dense precipitate of mitochondria carefully suspended in 0.5 ml of 0.25 M sucrose. Received a heavy suspension of mitochondria transferred into a test tube and kept on ice.

5.2. Getting preparation of rat liver mitochondria, specifically lunch is built on cytochrome C

40 ml of rat liver mitochondria (the protein concentration of 50 mg/ml) is placed in a hypotonic solution containing 0.01 M sucrose, 15 mm KCI. Incubated for 10 min in ice with stirring. Then centrifuged at 20000 g for 15 minutes the Precipitate resuspended in a small amount of 0.25 M sucrose using a homogenizer with a Teflon pestle. Then the drug is placed in a solution containing 150 mm KCI. Incubated for 10 min in ice with stirring and centrifuged at 20000 g for 15 minutes To the precipitate add a small amount of 0.25 M sucrose, homogenized, poured in aliquots of 1.5 ml of the resulting preparation is cryopreserved and stored in liquid nitrogen.

5.3. Measurement of succinate: cytochrome C reductase activity of rat liver mitochondria, specifically depleted by cytochrome C

Succinate: cytochrome C reductase activity measured in the recovery of cytochrome C spectrophotometrically at 550 nm. The sample volume of 2 ml of the incubation medium (0.15 M sucrose, 20 mm KCI, 20 mm Tris-HCI, pH 7.4, 5 mm NaN3), the preparation of mitochondria (9 μg/ml) and oxidized cytochrome C horse wild type or ACC (Sigma), or mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K. The concentration of oxidized cytochrome C horse wild type vary from 1 to 40 μm, mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or 8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, or ACC from 1 to 60 μm. The reaction starts with the addition of 10 mm succinate. Activity is expressed as µmol restored cytochrome C/min per 1 mg of protein of the mitochondria (figure 1).

Succinate: cytochrome C reductase activity of mitochondria in the presence of ACC is 8% of the activity in the presence of cytochrome C horse wild type. Succinate: cytochrome C reductase activity of mitochondria in the presence of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K is 3% of the activity in the presence of cytochrome C horse wild type (figure 1).

5.4. Measurement of cytochrome C oxidase activity of rat liver mitochondria, specifically depleted by cytochrome C

Cytochrome C oxidase activity was measured polarographically. The sample volume of 1.3 ml of the incubation medium (0.15 M sucrose, 20 mm KCI, 20 mm Tris-HCI, pH 7.4, 10 mm ascorbate), preparation of mitochondria (43 µg/ml) and oxidized cytochrome C horse wild type or ACC, or mutant cytochrome C K27E/E69K/K72E/K86E/K87E/E90K, or K8E/E62K/E69K/K72E/K86E/K87E, K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K. The concentration of oxidized cytochrome C horse wild-type or mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K, or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K, or ACC range from 1 to 90 μm. The reaction starts with the addition of 0.2 mm tetramethyl-para-phenylenediamine (TMPD). Activity is expressed as µmol of oxidized citehr the mA/min per 1 mg of protein of the mitochondria (figure 2).

Cytochrome C oxidase activity of mitochondria in the presence of ACC is 3% of the activity in the presence of cytochrome C horse wild type. Cytochrome C oxidase activity of mitochondria in the presence of mutant cytochrome C horse K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E, or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K not detected (figure 2).

A method of obtaining a recombinant mutant of cytochrome C horse, including the introduction of mutations CE/EC/CE/CE/CE/EC, or CE/EC/EC/CE/CE/CE, or CE/CE/EK/EK/CE/CE/CE/EC using the method of site-directed mutagenesis based on cyclic polymerase reactions, gene cytochrome C horse, being part of the plasmid DNA pBPCYCS/3, followed by transformation of Escherichia coli strain JM-109 obtained recombinant plasmid DNA, the expression and secretion of the target protein using cation exchange and adsorption chromatography.



 

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