C端序列的删除对非出血重组蛇毒纤溶酶的影响

Effect of Deletion of C-terminal Region on Nonhemorrhagic Recombinant Fibrin(ogen)olytic Metalloprotease

【目的】研究C端序列对非出血重组纤溶酶(rFⅡ)特异性、活性和热敏感性的影响。【方法】通过SOEingPCR方法构建删除C端序列的突变体,突变体和rFⅡ分别在P.pastoris中诱导表达。表达和纯化的蛋白用SDS-PAGE和Westerenblot进行鉴定。之后分析其生化特性。【结果】rFⅡ及突变体通过SDS-PAGE和Westerenblot得到证实。生化分析揭示:①突变体对显色底物N-(p-Tosyl)-Gly-Pro-Lys-pNA的催化效率(Kcat/Km)是rFⅡ的1.9倍;②突变体和rFⅡ对氧化的胰岛素B链拥有共同的优先裂解位点,可是在随后的裂解中开始展现差异;③突变体显示了更高的纤(原)活性;④热处理表明突变体相较rFⅡ对温度的增加更敏感;⑤通过圆二色谱测量,突变体的螺旋比例有所增加。【结论】删除的序列参与rFⅡ的特异性、活性和热敏感性,该序列可作为下一步优化的候选序列。

[Objective]To investigate the effect of C-terminal region on the specificity, activity, and heat-sensitivity of nonhemorrhagic recombinant fibrin(ogen)olytic metalloprotease (rFⅡ). [Method]SOEing PCR was used for constructing mutation of deleting the C-terminal region. The mutation and rFⅡ were induced and expressed in transformed P.pastoris cells respectively. The expressed and purified proteins were identified by SDS-PAGE and Western blot analysis. After that, biochemical characteristics were analyzed.[Result]The mutation and rFⅡ were confirmed by SDS-PAGE and Western blot analysis. Biochemical characteristics analysis revealed:(1) Mutation caused 1.9 fold increase of catalytic efficiency (Kcat/Km) against chromogenic substrate (N-(p-Tosyl)-Gly-Pro-Lys-pNA); (2)The mutation and rFⅡ shared common preferential cleavage site(12-13) of oxidized insulin-B chain while the difference started to display at secondary cleavage site; (3) Fibrin(ogn)olytic activity analysis revealed a higher activity in mutation. (4) Heat treatment indicated that mutation was more sensitive than rFⅡ to increase of temperature; (5) Furthermore, the ratio of helix of mutation increased by circular dichroism measurements.[Conclusion]The deleted sequence was involved in specificity, activity, and heat-sensitivity of rFⅡ and could be viewed as a candidate sequence for optimization.