嗜酸氧化亚铁硫杆菌半胱氨酸合成酶的表达、纯化及其性质鉴定

Expression,Purification and Characterization of a Cysteine Synthase from Acidithiobacillus ferrooxidans

半胱氨酸合成酶是半胱氨酸合成反应的关键限速酶,催化了半胱氨酸合成的最后一个步骤。以A.ferro-oxidans ATCC 23270基因组为模板,通过PCR扩增得到编码半胱氨酸合成酶(cysM)的基因,与原核表达载体PLM 1构建重组体,转入大肠杆菌DH5α中,测序正确后,加IPTG诱导表达,用一步亲和层析法纯化出了浓度和纯度都较高的半胱氨酸合成酶。由蛋白颜色和紫外分析,确定是以PLP辅基作为活性中心。表达产物进行SDS-PAGE分析,证实分子量为32 kD。

Cysteine synthase as a key enzyme involved in the pathway of the cysteine biosynthesis,can catalyze the last step of the cysteine biosynthesis forming L-cysteine and acetate from O-acetyl-L-serine（OAS）and sulfide.According to A.ferrooxidans ATCC 23270 genomes as template,the gene cysM which encodes cysteine synthase was cloned,and the fragment was linked into the prokaryotic expression vector PLM 1.Then the recombined expression plasmid was transduced into DH5α.After sequence was mensurated right,with the addition IPTG expressed in Escherichia coli,the soluble protein was purified by one-step affinity chromatography to apparent homogeneity.Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5′-phosphate（PLP）-containing protein.The molecular mass of the cysteine synthase was 32 kD determined by SDS-PAGE.