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

作　　者：秦春燕[1] 张旭[1] 陈谷[1] 

机构地区：[1]华南理工大学轻工与食品学院,广州510640

出　　处：《微生物学报》2012年第1期130-135,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30800609)~~

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。[ 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 Sl10862, the site-2 protease homologs from Synechocystis sp. PCC6803. [ Methods] Recombinant Slr0643 and Sl10862 were constructed and overexpressed in Escherichia coli BL21 （CE3）. Their protease activities were tested against/3-casein and then resolved on SDS-PAGE. [ Results] Results from caseinolytic assay indicated that Slr0643 and Sl10862 have proteolytic activity which is blocked by o-phenanthroline, a metalloprotease inhibitor. These metalloprotease activity of Slr0643 and Sl10862 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.

关 键 词：集胞藻PCC6803 S2P金属蛋白酶 Slr0643 Sll0862 原核表达 纯化 体外酶活鉴定 

分 类 号：Q936[生物学—微生物学]