烟草蚀刻病毒
蛋白酶
突变体
劈理(地质)
大肠杆菌
生物化学
融合蛋白
圆二色性
溶解度
化学
氨基酸
肽
肽序列
体内
分子生物学
酶
生物
重组DNA
病毒
基因
植物病毒
马铃薯Y病毒
病毒学
有机化学
古生物学
生物技术
断裂(地质)
作者
Lingling Wei,Xueyan Cai,Qi ZhenGuo,Liang Rong,Beijiu Cheng,Jun Fan
标识
DOI:10.1016/j.pep.2012.03.011
摘要
Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.
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