生物化学
蛋白质水解
融合蛋白
化学
蛋白酶
氧化磷酸化
大肠杆菌
氧化还原
溶解度
热稳定性
作者
Enkhtuya Bayar,Yuanyuan Ren,Yinghua Chen,Yafang Hu,Shuncheng Zhang,Xuelian Yu,Jun Fan
出处
期刊:Journal of Microbiology and Biotechnology
[Journal of Microbiology and Biotechnology]
日期:2021-12-28
卷期号:31 (12): 1732-1740
标识
DOI:10.4014/jmb.2106.06075
摘要
Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.
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