SrtA蛋白在大肠杆菌中的高效表达与活性鉴定

按大肠杆菌偏好优化密码子并合成高活性突变型srtA基因，构建Trx蛋白共表达载体pTIG-srtA，转入大肠杆菌BL21（DE3）后低温诱导表达，通过Ni2+柱亲和层析纯化SrtA蛋白，连接LH_N-LPETG和G-H_C蛋白检测SrtA蛋白活性。菌液PCR鉴定和测定序列比对结果显示，构建pTIG-srtA载体成功；SDS-PAGE及Western Blot分析结果显示，在大肠杆菌中实现了SrtA蛋白的可溶性高表达，Ni2+柱亲和层析后得到纯度大于95%的SrtA蛋白，LH_N-LPETG和G-H_C蛋白成功连接表明，原核系统表达纯化的SrtA蛋白具有良好的转肽酶活性。

High-efficiency Expression and Activity Identification of SrtA Protein in Escherichia coli

Optimize codons according to E. coli preference and synthesize highly active mutant srtA geneto construct the Trx protein co-expression vector pTIG-srtA. Then it was transformed into E. coli BL21(DE3) and induced expression at low temperature, and the SrtA protein was purified by Ni2+ column affinity chromatography, and connected LH_N-LPETG and G-H_C protein to detect SrtA protein activity. The results of bacterial liquid PCR identification and determination of sequence comparison showed that the pTIG-srtA plasmid was successfully constructed. The results of SDS-PAGE and Western Blot analysis showed that high soluble expression of SrtA protein was achieved in E. coli, Ni2+ column affinity chromatography obtains SrtA protein with a purity greater than 95%. The successful connection of LH_N-LPETG and G-H_C protein indicated that the SrtA protein expressed and purified by the prokaryotic system had good transpeptidase activity.