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 Ni2+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,Ni2+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.
出处 《食品工业科技》 CAS 北大核心 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)。
关键词 SrtA 密码子优化 酶连接法 A型肉毒毒素 Sortase A codon optimization enzyme ligation method botulinum neurotoxin A
