Efficiency of <i>Escherichia coli</i> and <i>Bacillus subtilis</i> Expression Systems for Production of Binase Mutants

Abstract

Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while at high concentrations it causes genotoxic effects. The use of mutants with reduced catalytic activity preserving the antitumor properties of the native enzyme could reduce the toxic side effects of the enzyme. Here, mutant forms of binase with Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli and Bacillus subtilis-based expression systems demonstrated the feasibility of using a bacilli-based heterologous system for production binase mutants. Binase mutants with reduced catalytic activity were isolated and purified with ion exchange chromatography in a homogeneous state with 25 mg/L yield. The catalytic properties of obtained mutants toward natural RNA-substrates in comparison with those for native binase were analyzed. The catalytic activity of the Lys26Ala and His101Glu mutants was 11 and 0.02%, respectively. It was found that the Lys26Ala mutant as well as the native binase exhibits selective cytotoxicity toward A549, BT-20 and HuTu 80 tumor cell lines, without causing toxic effects toward normal WI-38 cells. The mutant His101Glu did not exhibit cytotoxicity.