Abstract：
Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis-based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity.
摘要：
短小芽孢杆菌核糖核酸酶（binase）具有细胞毒性和溶瘤特性，同时在高浓度下引起基因毒性作用。具有降低的催化活性并保留天然酶的抗肿瘤特性的突变体可以发挥较低的毒副作用。通过定点诱变获得具有Lys26Ala和His101Glu单取代的突变体结合酶形式。基于大肠杆菌和枯草芽孢杆菌的表达系统的比较分析表明，后者更适合用于生产binase突变体。通过离子交换色谱法分离和纯化具有降低的催化活性的binase突变体。最大产量为25mg/L。与天然binase相比，突变体对天然RNA底物的催化活性估计为11％和0.02％，分别。像天然的Binase一样，发现Lys26Ala突变体对A549，BT-20和HuTu80肿瘤细胞系具有细胞毒性，但不显著影响正常WI-38细胞。His101Glu突变体未显示细胞毒性。
