Journal of Microbiology and Biotechnology

  • 제31권12호
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  • Pages.1732-1740
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  • 2021
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

  • Bayar, Enkhtuya (School of Life Science, Anhui Agricultural University) ;
  • Ren, Yuanyuan (School of Life Science, Anhui Agricultural University) ;
  • Chen, Yinghua (School of Life Science, Anhui Agricultural University) ;
  • Hu, Yafang (School of Life Science, Anhui Agricultural University) ;
  • Zhang, Shuncheng (School of Life Science, Anhui Agricultural University) ;
  • Yu, Xuelian (School of Life Science, Anhui Agricultural University) ;
  • Fan, Jun (School of Life Science, Anhui Agricultural University)
  • 투고 : 2021.06.25
  • 심사 : 2021.09.08
  • 발행 : 2021.12.28
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초록

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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과제정보

This study was supported by the Anhui Educational Committee Foundation (KJ2020A0113).

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