Identification of Functional Regions in the Empha

Authors: M V Pokrovskaya S S Aleksandrova V S Pokrovsky A V Veselovsky D V Grishin O Yu Abakumova O V Podobed A A Mishin D D Zhdanov N N Sokolov

Publish Date: 2014/11/05

Volume: 57, Issue: 3, Pages: 251-264

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Abstract

Sitedirected mutagenesis of Rhodospirillum rubrum lasparaginase RrA was performed in order to identify sites of the protein molecule important for its therapeutic and physicochemical properties Ten multipoint mutant genes were obtained and five recombinant RrA variants were expressed in E coli BL21DE3 cells and isolated as functionally active highly purified proteins Protein purification was performed using QSepharose and DEAEToyopearl chromatography Overall yield of the active enzymes was 70–80  their specific activity at pH 74 and 37 °C varied of 140–210 U/mg lGlutaminase activity did not exceed 001  of lasparaginase activity All RrA mutants showed maximum enzyme activity at pH 93–95 and 53–58 °C Km and Vmax values for lasparagine were evaluated for all mutants Mutations G86P D88H M90K RrAH G121L D123A RrАI caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions Removal of four residues from Cterminal area of the enzyme RrAK resulted in the enzyme instability Mutations D60K F61LRrАD and R118H G120RRrАJ led to the improvement of kinetic parameters and enzyme stabilization Substitutions E149R V150P RrАB improved antineoplastic and cytotoxic activity of the RrA A64V E67K substitutions especially in combination with E149R V150P RrАE considerably destabilized recombinant enzymeWe express our sincere gratitude to Dr Mikhail A Eldarov Centre “Bioengineering” RAS Moscow Russia for his participation in the discussion constructive suggestions useful critique of this research work as well as for help with translation of this article Dr Vasiliy N Lazarev’s Scientific Research Institute of Physical–Chemical Medicine Moscow Russia valuable support and assistance in DNA sequencing and Prof Ekaterina Kolesanova’s Orekhovich Institute of Biomedical Chemistry Moscow Russia help with protein oligomerisation studies are greatly acknowledged

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