Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

Bayar Enkhtuya; Ren Yuanyuan; Chen Yinghua; Hu Yafang; Zhang Shuncheng; Yu Xuelian; Fan Jun

doi:10.4014/jmb.2106.06075

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자료유형: 학술저널

저자정보: Bayar Enkhtuya (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Ren Yuanyuan (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Chen Yinghua (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Hu Yafang (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Zhang Shuncheng (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Yu Xuelian (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China) Fan Jun (School of Life Science Anhui Agricultural University Hefei Anhui 230036 P.R. China)

저널정보: 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제31권 제12호

발행연도: 2021.12

수록면: 1,732 - 1,740 (9page)

DOI: 10.4014/jmb.2106.06075

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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.

#TEVp #mutation #cysteine residues #oxidative stability #tag removal #Escherichia coli

참고문헌 (35)

참고문헌 신청

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Cesaratto F / 2016 / Tobacco Etch Virus protease : a shortcut across biotechnologies / J. Biotechnol 231:239~249

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Kapust RB / 2001 / Tobacco etch virus protease : mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency / Protein Eng :14

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