大肠杆菌重组乳链菌肽抗性蛋白(NSR)的表达纯化及其功能分析  

Purification and Functional Analysis of Recombinant Nisin Resistance Protein (NSR) Expressed in Escherichia coli

作　　者：刘家乐[1,2] 孙志增[2] 刘一苇[2] 高学玲[1] 钟瑾[2] 

机构地区：[1]安徽农业大学茶与食品科技学院,安徽合肥230036 [2]中国科学院微生物研究所,北京100101

出　　处：《微生物学通报》2009年第10期1519-1525,共7页Microbiology China

基　　金：国家863计划项目(No.2006AA10Z319);中国科学院知识创新重要方向项目(No.KSCX2-YW-G-016)

摘　　要：乳链菌肽(Nisin)是由某些乳酸菌产生的一种阳离子抗菌肽,而Nisin抗性蛋白(Nisin resistance protein,NSR)的表达则使一些非Nisin产生菌获得Nisin抗性。为深入探索NSR的作用机制,本研究在大肠杆菌中表达了去除N末端38个氨基酸残基的NSR(NSRΔ38)与GST的融合蛋白GST-NSRΔ38。通过谷胱甘肽(GSH)亲和层析和GST标签的切除后,得到纯化的NSRΔ38,并测定了该蛋白可能的nisin降解活性。反应产物的抑菌活性测定结果表明被NSRΔ38作用后的Nisin丧失了抑菌活性,而反相高效液相层析(RP-HPLC)分析结果进一步表明这是由NSRΔ38具备Nisin降解活性所造成的。本研究为NSR功能的深入研究奠定了工作基础。Nisin is a cationic antimicrobial peptide produced by some lactic acid bacteria. However, expression of nisin resistance protein （NSR） could confer nisin resistance on some non-nisin-producing Lactococcus lactis. To deeply elucidate molecular mechanism underlying NSR-mediated nisin resistance, an NSR mutant with N-terminal 38 amino acid residues deleted （NSRA38） was overexpressed in Escherichia coli by fusion with GST. Purified NSRA38 was obtained through glutathione （GSH） affinity chromatography followed by cleavage of GST tag. Putative proteolytic activity of NSRA38 was determined in vitro against nisin Antimicrobial activity analysis revealed that nisin lost its bactericidal activity after incubation with NSRA38. Further reversed-phase high performance liquid chromatography （RP-HPLC） analysis indicated that NSRA38 displayed proteolytic activity against nisin, thus inactivating the antimicrobial peptide. The current study paves the way for in-depth functional studies on NSR.

关 键 词：乳链菌肽 乳链菌肽抗性蛋白 末端特异性蛋白酶 GST融合蛋白 

分 类 号：Q936[生物学—微生物学]