INVESTIGATION OF BACILLAR EXTRACELLULAR BARNASE AND BINASE RIBINUCLEASES BY A SITE-SPECIFIC MUTAGENESIS METHOD

Abstract

The influence of the amino acid sequence in the fixation site of the substrate 3'-foundation on the specifics of the intracellular, cyclic and bacillar barnase and binese ribonucleases in the hydrolysis processes of the polyribonucleotide substrates has been investigated. The high-effective system for expression of the barnase and binase in E. coli based on using power strong-regulated stimulator P*00r of the phage lambda under control of the thermosensitive expressor has been developed. The effect of depending quantity of the protein eluted from supine-phased (SP) sorbent upon PH of the movable phase has been discovered for a number of the bacillar extracellular ribonecleases. The hypothesis explaining the discovered effect has been proposed, and the experimental confirmation of this hypothesis has been obtained. The two-staged scheme for cleaning of the intracellular bacillar ribonucleases (RNC) and their mutant derivatives based on their properties in the conditions of SP-high-effective liquid chromatography has been developed. The mutant derivatives of the barnase and binase in the field of the "learning loop" have been obtained. Their catalytic properties in the hydrolysis reactions of the purine homopolyribonucleotides have been studied. It has been manifested that the contribution of the specific fixation of the substrate foundation in the "learning loop" into the fixation constant value is isufficient. It has been shown that the substitution of the "learning loop" of the guanylo-preferable RNC Bi on the "learning loop" of the guanylo-specific RNC Sa doesn't lead to the total change of the mutant protein guanylo-specifics in the hydrolysis reactions of the homopolyribonucleodides. The results can be used in the Institute of Molecular Biology (Russian Academy of Sciences), All-Russian Institute of Genetics and Industrial Microorganisms, Institute of Molecular Genetics (Russian Academy of Sciences) and Institute of General Genetics (Russian Academy of Sciences)Available from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio