【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sufS-pET28a和sufEpGEX-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SufE1.0~2.5 h,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-Atf-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72 h.更多还原

【Abstract】 Iron-sulfur clusters play important roles in electron transport,redox and nonredox catalysis and regulation of gene expression.SufS and SufE were separately amplified by the PCR using the Thermus thermophiles HB8 chromosomal DNA as a template.These two genes have been ligated into pET28 a and pGEX-6P-1 vectors,respectively,generating recombination plasmid sufS /pET28 a 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 SufE 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 activity was kept at 60℃ for 72 hours.更多还原