SrtA蛋白在大肠杆菌中的高效表达与活性鉴定  

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

作　　者：张秀娟[1] 于海威 刘栋琦 赵晋彤 熊向华 ZHANG Xiujuan;YU Haiwei;LIU Dongqi;ZHAO Jintong;XIONG Xianghua(School of Pharmacy,Harbin University of Commerce,Harbin 150076,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]哈尔滨商业大学药学院,黑龙江哈尔滨150076 [2]军事科学院军事医学研究院生物工程研究所,北京100071

出　　处：《食品工业科技》2021年第10期83-88,共6页Science and Technology of Food Industry

基　　金：国家科技重大专项资助项目(2018X09J18105-003)。

摘　　要：按大肠杆菌偏好优化密码子并合成高活性突变型srt A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Ni^(2+)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,Ni^(2+)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。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 Ni^(2+)column affinity chromatography,and connected LHN-LPETG and G-HC 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,Ni^(2+)column affinity chromatography obtains SrtA protein with a purity greater than 95%.The successful connection of LHN-LPETG and G-HC protein indicated that the SrtA protein expressed and purified by the prokaryotic system had good transpeptidase activity.

关 键 词：SrtA 密码子优化 酶连接法 A型肉毒毒素 

分 类 号：TS201.2[轻工技术与工程—食品科学]