[Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila]. / Poluchenie i kharakteristika novogo mutantnogo gomologa khemotaksisnogo belka CheY iz gipertermofil'nogo anaérobnogo mikroorganizma Thermotoga naphthophila.

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.

[Construction and characterization of a recombinant mutant homolog of the CheW protein from Thermotoga petrophila RKU-1]. / Poluchenie i kharakteristika rekombinantnogo mutantnogo gomologa belka CheW iz gipertermofil'nogo mikroorganizma Thermotoga petrophila RKU-1.

In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.

[Prediction of protein thermostability from their primary structure: the current state and development factors]. / Prognozirovanie termostabil'nosti belkov po ikh pervichnoi strukture: sovremennoe sostoianie i faktory razvitiia.

The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.

[Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase]. / Podavlenie aktivnosti telomerazy leikoznykh kletok mutantymi formami L-asparaginazy Rhodospirillum rubrum.

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

Creation of a producent, optimization of expression, and purification of recombinant Yersinia pseudotuberculosis L-asparaginase.

Recombinant E. coli strain producing Y. pseudotuberculosis Q66CJ2 (YpA) L-asparaginase II was created. Gene ansB homologue encoding Y. pseudotuberculosis IP 32953 L-asparaginase precursor was synthesized. The gene was cloned in pBad24 expression vector and expressed in E. coli BL21 (DE3) strain. Optimal conditions for the producer strain culturing were selected. An effective method for isolation and purification of the enzyme by two-staged column chromatography was developed.