集胞藻PCC6803中S2P同源蛋白Slr0643及Sll0862 金属蛋白酶活性的体外鉴定

Characterization of metalloprotease of Slr0643 and Sll0862，the S2P homologs from Synechocystis sp．PCC6803

摘要:

摘要:【目的】金属蛋白酶S2P 在细菌中通过在膜切割转录调控因子、释放δ因子参与胁迫响应是跨膜信号转导的保守机制，但蓝细菌中S2P的功能还未被鉴定，故我们考察集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862的金属蛋白酶活性。【方法】以pET-30b(+)为载体，分别构建重组质粒pF0643和pF0862，在大肠杆菌BL21(CE3)中诱导表达并纯化Slr0643及Sll0862蛋白，以β-酪蛋白为底物检测重组蛋白的酶活性。【结果】体外酶活实验显示重组表达的Slr0643及Sll0862蛋白有内切蛋白酶活性，且其活性受金属螯合剂o-phenanthroline的抑制。体外酶活的鉴定结果为进一步研究Slr0643 和Sll0862 的体内酶活和生物学功能奠定了基础。【结论】集胞藻PCC6803 中的S2P 同源蛋白Slr0643 及Sll0862 具有金属蛋白酶活性。

Abstract:

Abstract:［Objective］It is a conserved mechanism in bacteria that metalloprotease site-2 protease (S2P) cleaves transmembrane anti-sigma factor to release sequestered sigma factor in response to extracytoplasmic stress． However，the function of site-2 protease homologs in cyanobacteria remains elusive，so we investigated the metalloprotease activity of Slr0643 and Sll0862，the site-2 protease homologs from Synechocystis sp． PCC6803. ［Methods］Recombinant Slr0643 and Sll0862 were constructed and overexpressed in Escherichia coli BL21 (CE3)．Their protease activities were tested against β-casein and then resolved on SDS-PAGE．［Results］Results from caseinolytic assay indicated that Slr0643 and Sll0862 have proteolytic activity which is blocked by o-phenanthroline，a metalloprotease inhibitor． These metalloprotease activity of Slr0643 and Sll0862 in vitro provide the foundation for futher analysis of their substrates in vivo．［Conclusion］The site-2 protease homologs in Synechocystis sp． PCC6803 have metalloprotease activity．