荚膜红细菌DSM1710中硫化物醌还原酶基因的隆、表达及活性分析

摘要

为获得有活性的硫化物醌还原酶(sulfide-quinone reductase,SQR)并研究其功能,试验利用常规PCR方法从荚膜红细菌(Rhodobacter ca psulatus)中克隆了SQR基因全长CDS区1284 bp;成功构建了SQR基因的原核表达载体pRSETA-SQR,并转入大肠杆菌中进行诱导表达.SDS-PAGE和Western blotting结果显示,SQR融合蛋白成功表达,在37℃条件下,0.4 mmoL/L IPTG诱导表达6h,50 ku的SQR融合蛋白表达量最高.可溶性分析结果表明,SQR重组蛋白以包涵体形式存在,经过纯化、复性后得到较高纯度的SQR活性蛋白.蛋白活性分析结果表明,该酶具有明显的消化底物decyl-UQ的能力,测定其酶活Km值约为4.%To analyze the characteristics and function,we cloned the complete 1284 bp CDS of the sulfide quinone reductase (SQR) gene from Rhodobacter capsulatus DSM1710. Then the prokaryotic expression vector pRSETA-SQR for the SQR gene was successfully constructed. Expression of pRSETA-SQR fusion protein with a molecular mass of approximately 50 ku in Escherichia coli BL2KDE3) induced by IPTG was confirmed by SDS-PAGE and Western blotting. The SDS-PAGE result indicated that with the induction of 0. 4 mmol/L IPTG for 6 h at 37 ℃ ,the fusion protein of the SQR was expressed well. The fusion protein was expressed as inclusion body. The purification and renaturation results showed that the SQR recombinant protein had the bioactivity and could digest the substrate decylubiquinone (decyl-UQ). The enzyme activity was measured with Km value of about four.