[Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis].

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Evaluation of the Effectiveness of Metabolites of Bacterial Strains Bacillus thuringiensis against Human Influenza Virus A/Aichi/2/68 (H3N2) In Vitro and In Vivo.

The morphological and physiological characteristics of Bacillus thuringiensis strains were analyzed and conditions for obtaining culture fluid with maximum yield of secreted RNases were determined. Zymographic analysis showed that culture fluid of B. thuringiensis strains along with low-molecular-weight (15-20 kDa) RNases contained enzymes with a molecular weight ~55 kDa and their content depended on the duration and conditions of culturing. Preparations based on B. thuringiensis culture fluid were effective against human influenza virus A/Aichi/2/68 (H3N2). In experiments on mice infected with 10 LD50 influenza virus strain A/Aichi/2/68 (H3N2), we selected effective variants of preparations based on culture fluid of B. thuringiensi strains for preventive administration that provided reliable protection of infected animals (protection coefficient 50%), close to that of the reference drug Tamiflu.

DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin's Inhibitory Effect.

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30 kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-70 °C, and both were inactivated via heating at 95 °C for 15 min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.

[Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum].

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.

[Cloning and expression of the gene of chymotrypsin-like protease of human kallikrein-7 in Escherichia coli and isolation of recombinant protein].

A sample of the human cDNA was used to amplify the segment encoding biosynthesis of chymotrypsin-like protease of kallikrein-7 and its cloning into the expressing plasmid pET23a(+). Biosynthesis of KLK-7 in transformed E. coil BL21(DE3) cells was accompanied by formation of insoluble inclusion bodies. The recombinant KLK-7 was extracted from the inclusion bodies using 7 M urea in the presence of 2-mercaptoethanol. The extracted recombinant KLK-7 was purified using methods of metal-chelate and ion-exchange chromatography, converted into a soluble form, and used for preparing monospecific antiserum.

[The determination of lipopolysaccharide admixtures in protein preparations]. / Opredelenie primesei lipopolisakharidov v preparatakh belkov.

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.

[Design of an expression vector for delivery of in vivo recombinant biologically active proteins. 1. Synthesis of the antigenic determinant of HIV-1 gp120 and gp41 proteins in enterobacteria]. / Konstruirovanie ékspressionnogo plazmidnogo vektora dlia osushchestvleniia dostavki in vivo rekombinantnykh biologicheski aktivnykh belkov. 1. Sintez antigennykh determinant belkov gp120 i gp41 VICh-1 v énterobakteriiakh.

High-level expression vector pAZ was constructed for in vivo delivery of bioactive recombinant proteins, antigenic determinants, among other things. This vector meets the requirements to construction of recombinant bacteria as live oral carriers. It has a strong constitutive promoter, high stability in E.coli and vaccine strain Salmonella cells, and, moreover, encodes in addition for the marker protein (beta-galactosidase) which will later help follow up the fate of bacterial carriers and their interactions in the microorganism. Several recombinant plasmids encoding for beta-galactosidase variants with insertions of short fragments of HIV-1 gp41 and gp120 proteins, which were previously shown to be antigenic determinants, have been constructed on the basis of pAZ. E.coli and vaccine strain Salmonella cells were transformed by recombinant plasmids. To a considerable extent the level of hybrid protein synthesis depends on the structure of the antigenic determinant inserted. The maximal level of synthesis in E.coli is 16%. This hybrid protein could be isolated and purified (up to 90%) with the yield of 4 to 6 mg/g of wet cells. Almost all the hybrid proteins were immunologically reactive, as shown by ELISA with nonfractionated lysates and purified hybrids. In both strains in vitro stability of the vector and recombinant plasmids was at least 90% after 10 passages (about 140 generations) under random conditions. This paper sums up the first stage of construction of recombinant bacteria as live oral carriers.

[Effectiveness of the promoter Ptac' in vitro and in mini-cells]. / Izuchenie éffektivnosti promotora Ptac' in vitro i v mini-kletkakh.

The efficiency of hybrid promotor Ptac', comprising a synthetic trp-promoter and lacUV5 "-10" sequence, was studied. By means of electrophoresis and hybridization of RNA-products obtained in vitro and in minicells, with promotor-containing plasmids; the hybrid promotor was found to be 6 times and 3-4 times as efficient as trp and lacUV5 promoters, respectively.

[Expression of the gene for hybrid beta-lactamase pBR322 with C-terminal fragment containing tuftsin (Thr-Lys-Pro-Arg)]. / Izuchenie ékspressii gena gibridnoi beta-laktamazy pBR322 s C-kontsevym fragmentom, soderzhashchim taftsin (Thr-Lys-Pro-Arg).

A hybrid beta-lactamase gene with a synthetic tuftsin-coding DNA fragment inserted at the Pst I-site of pBR322 plasmid has been obtained and its expression has been studied. Radioactive amino acids have been used to show that in E. coli chi 925 minicells up to 30% of newly synthesized chimeric protein is secreted into periplasm providing the tuftsin transport. After hybrid protein cleavage with CNBr, tuftsin has been isolated using ion-exchange and thin-layer chromatography.

[Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method]. / Izuchenie strukturnoi organizatsii dvuspiral'noi RNK killernykh drozhzhei Sacharomyces cerevisiae metodom termicheskoi denaturatsii.

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.

[Circular transcription map of the plasmid pBR322]. / Kol'tsevaia transkriptsionnaia karta plazmidy pBR322.

The plasmid pBR322 transcription in the isolated E. coli DNA-dependent RNA-polymerase system was studied. Transcription regions as well as transcripts orientation were defined using both the technique of "criss-cross" hybridization and the annealing of RNA-products with L- and H-strands of plasmid. Summing up obtained data together with available data on promoter localization a circular transcription map of plasmid pBR322 was constructed. Effects of heparin, ion strength and E. coli S-30 system on in vitro transcripts were also studied. The 110-long RNA transcript synthesized in Ori region of pBR322 was found to be the most sensitive to all these factors. RNA-transcripts obtained in in vitro system are able to direct protein synthesis in cell-free S-30 system.

[Laboratory and clinical study of biological activity of poly G-poly C complex]. / Laboratornoe i klinicheskoe issledovanie biologicheskoi aktivnosti kompleksa poli(G).poli(Ts).

Poly(G).poly(C) inoculated intravenously to mice in a dose of 100 microgram induced interferon in the blood in amounts comparable to those induced by poly(I).poly (C). In contrast to rapid accumulation (within 2 hours after induction) and rapid disappearance of interferon in response to poly(I).poly(C) inoculation, the interferon induced by poly(G).poly(C) reached the maximum titer by 6 hours and remained at a high level for 24 hours after inoculation. When given to human volunteers intranasally in a dose of 6 mg, the poly(G).poly(C) complex induced interferon in the blood serum in 70% of the subjects in a titer of 85 units/ml within 24 hours.