嗜热菌HB8 Fe-S簇生物合成相关酶SufS与SufE重组酶的体外复合体功能  

Function of the in vitro Recombinant SufS-SufE Complex in the Fe-S Cluster Biosynthesis Pathway from the Thermus thermophilus HB8

作　　者：张宗棋[1] 田甜[1] 王妮莎[1] 刘晓晴[1] 

机构地区：[1]首都师范大学生命科学学院,北京100048

出　　处：《中国生物化学与分子生物学报》2014年第5期496-502,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助(No.31070067)项目~~

摘　　要：Fe-S簇在细胞的生物学过程中发挥重要作用，参与电子传递和基因调节等过程．以嗜热菌HB8基因组为模板，通过PCR扩增获得Fe—S簇SUF系统生物合成途径中的sufS与sufE两个基因．将目的基因分别连接到表达载体pET28a和pGEX-6P-1上，构建重组质粒,CS-pET28a和sufE-pGEX-6P-1．重组质粒转化E．coli BL21（DE3）菌株进行蛋白质表达及亲和层析纯化．研究证明，sufS具有脱硫酶活性，而SufE的存在可增强其活性．体外重组实验表明，在60℃孵育SufS和SufE1．0～2．5h，其复合体脱硫酶活性约是单体sufS的2倍，但两者相互作用时间短暂．若通过交联剂Mts—Aft-Biotin使SufS与sufE共价连接，此时SufsE酶活性是SufS的6倍左右，并可持续72h．Iron-sulfur clusters play important roles in electron transport, redox and nonredox catalysis and regulation of gene expression. SufS and SurE were separately amplified by the PCR using the Thermus thermophiles HB8 chromosomal DNA as a template. These two genes have been ligated into pET28a and pGEX-6P-1 vectors,respectively, generating recombination plasmid sufS/pET28a and sufE/pGEX-6P-1. These recombinant plasmids were expressed in E. coli BL21 （DE3）, respectively. The proteins were purified by affinity chromatography and gel filtration. The recombination measurement showed that the activity of SufSE complex was about 2-fold more than that of the SufS monomer. And the activity of SufSE complex was kept at 60℃ for 2.5 hours. Covalently-bonding of the SufS and SurE via cross-linking agent Mts-Atf-Biotin has been completed. It showed that the activity of SufSE complex was about 6-fold higher than that of the SufS. and the activitv was kent at 60℃ for 72 haurs.

关 键 词：嗜热菌HB8 FE S簇 交联剂 重组 

分 类 号：Q55[生物学—生物化学]