嗜酸氧化亚铁硫杆菌硫氰酸酶的表达、纯化及其性质鉴定

Expression, Purification, and Characterization of Rhodanese from Acidithiobacillus ferrooxidans

目的:嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)硫氰酸酶是硫代谢中极其重要的酶,其作用主要包括氰化物的解毒,铁-硫蛋白的合成,以及硫胺、硫尿苷或烟碱乙酸胆碱的生物合成,硫氰酸酶的研究对揭示生物冶金机理具有重要的推动作用。方法:以A.ferrooxidans ATCC23270基因组为模板设计引物,通过PCR扩增得到编码硫氰酸酶的基因,目的基因片段与原核表达载体PLM1构建重组体,然后转入大肠杆菌(Escherichia coli,E.coli)DH5a感受态中,基因测序正确后,重组质粒再转入E.coliBL21感受态中,加IPTG诱导蛋白表达,用一步亲和层析法纯化出浓度和纯度都较高的硫氰酸酶。结果:SDS-PAGE分析证实蛋白分子量为21kD,紫外可见光分析,确定硫氰酸酶中含有铁硫簇,酶活测定发现重组硫氰酸酶在体外不具有酶活性,可能与酶反应条件及信号肽的切断有关。结论:体外成功克隆,表达,纯化出重组体硫氰酸酶,其基本性质也得到阐述。

Objective： Rhodanese is an important enzyme involved in the sulfur metabolize in Acidithiobacillus ferrooxidans. Proposed functions for rhodanese include cyanide detoxification, formation of prosthetic groups in iron-sulfur cluster proteins, and sulfur transfer for thiamine, thiouridine or molybdopterin biosynthesis. Research on rhodanese can help to explain the mechanisms of Bioleaching. Methods： The gene which encodes rhodanese was identified in whole genome ofA. ferrooxidans ATCC23270 and cloned. Then the fragment was linked into the prokaryotic expression victor PLM1. The recombined expression plasmid was transduced into Escherichia coli DH5a. After sequence being identified to be right, then the recombined expression plasmid was transduced into Escherichia coli BL21. The protein was expressed in the presence of IPTG, the soluble protein was purified by one-step affinity chromatography. Results： The molecular mass of rhodanese was 21kD confirmed by SDS-PAGE, UV-scanning determined the presence of iron-sulfur clusters, The lack of activity detection for rhodanese in vitro in our assays may be due to the reaction conditions or the lack of signal peptide. Conclusion： The recombinant rhodanese is successfully cloned, expressed and purified in vitro.Its essential characters have been described.