Covalent and non-covalent strategies for the immobilization of Tobacco Etch Virus protease (TEVp) on superparamagnetic nanoparticles

2020 ◽  
Vol 322 ◽  
pp. 1-9 ◽  
Author(s):  
Jessica L. Norris ◽  
Tulsi Patel ◽  
Anvesh K.R. Dasari ◽  
Thomas A. Cope ◽  
Kwang Hun Lim ◽  
...  
Keyword(s):  
Tobacco Etch Virus ◽  
Superparamagnetic Nanoparticles ◽  
Tobacco Etch Virus Protease

Exploring the Mutations on Improving Oxidative Stability of Tobacco Etch Virus Protease for Tag-removal in Refolding of Two Disulfide-rich Proteins

2021 ◽  
Author(s):  
Jun Fan ◽  
Enkhtuya Bayar ◽  
Yuanyuan Ren ◽  
Yafang Hu ◽  
Yinghua Chen ◽  
...  
Keyword(s):  
Oxidative Stability ◽  
Disulfide Bonds ◽  
Redox State ◽  
Protein Solubility ◽  
Tobacco Etch Virus ◽  
Tobacco Etch Virus Protease ◽  
Amorphous Cellulose ◽  
E Coli ◽  
Cleavage Activity ◽  
Cellulose Binding

Abstract Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp shows less oxidative stability, which limits its application under the oxidized redox state to facilitate disulfide bonds formation for refolding disulfide-bonded proteins. Previously, we combined six mutations into the TEVp to generate the TEVp5M for obviously increasing the protein solubility and decreasing the auto-cleavage. In this work, we introduced and combined C19S, C110S and C130S mutations into the TEVp5M to generate seven variants, analyzed protein solubility and the cleavage activity of the constructs in each of three E. coli strains including BL21(DE3), BL21(DE3)pLys, and Rossetta(DE3), and those of the optimized soluble variants in the oxidative cytoplasm of Origami(DE3) under the same induction conditions. The results suggested that desirable protein solubility, cleavage activity and oxidative stability are not combined. Unlike that of the C19S, introduction of the C110S and/or C130S less affected protein solubility but increased tolerance to the oxidative redox state. Use of the TEVp5MC110S/C130S variant, the refolded disulfide-rich bovine enteropeptidase or maize peroxidase was released via cleaving the sequence between the target protein and the cellulose-binding module bound to regenerated amorphous cellulose.

Crystal Structure of Tobacco Etch Virus Protease Shows the Protein C Terminus Bound within the Active Site

2005 ◽  
Vol 350 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Christine M. Nunn ◽  
Mark Jeeves ◽  
Matthew J. Cliff ◽  
Gillian T. Urquhart ◽  
Roger R. George ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Protein C ◽  
Tobacco Etch Virus ◽  
Tobacco Etch Virus Protease ◽  
C Terminus

scholarly journals Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16136 ◽  
Author(s):  
George Kostallas ◽  
Per-Åke Löfdahl ◽  
Patrik Samuelson
Keyword(s):  
Tobacco Etch Virus ◽  
Tobacco Etch Virus Protease ◽  
Whole Cell ◽  
Cell Assay

scholarly journals Covalent and Non-Covalent Strategies for the Immobilization of Tobacco Etch Virus (TEV) Protease on Superparamagnetic Nanoparticles

2019 ◽  
Author(s):  
Jessica L. Norris ◽  
Tulsi Patel ◽  
Anvesh K. R. Dasari ◽  
Thomas A. Cope ◽  
Kwang Hun Lim ◽  
...  
Keyword(s):  
Cost Saving ◽  
Fusion Proteins ◽  
Life Sciences ◽  
Tobacco Etch Virus ◽  
Superparamagnetic Nanoparticles ◽  
Tev Protease ◽  
Enzyme Retention

<div>TEV protease fusion proteins were prepared and immobilized on superparamagnetic nanoparticles (SPIONs). Non-covalent (avidin-biotin) and covalent (HALOtag-chloroalkane) strategies were explored for TEV immobilization. HALOtag immobilized TEV protease provided enhance performance in both enzyme retention and reusability. SPION-immobilized proteases could be a convenient and cost-saving tool in the life sciences</div>

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