scholarly journals Design of a new protease inhibitor by the manipulation of the bait region of α2-macroglobulin: inhibition of the tobacco etch virus protease by mutant α2-macroglobulin

1995 ◽  
Vol 312 (1) ◽  
pp. 191-195 ◽  
Author(s):  
L Van Rompaey ◽  
P Proost ◽  
H Van den Berghe ◽  
P Marynen
Keyword(s):  
Expression System ◽  
Tobacco Etch Virus ◽  
Tobacco Etch Virus Protease ◽  
Tev Protease ◽  
Cleavage Specificity ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Specific Protease ◽  
Inhibitory Spectrum ◽  
Primary Cleavage

Human alpha 2-macroglobulin (alpha 2M) inhibits a broad spectrum of proteases by changing its conformation and physically confining the enzyme. The inhibitory spectrum of alpha 2M is defined by a stretch of 39 amino acids, the bait region, located near the middle of the alpha 2M monomers. To investigate whether a new inhibitory specificity can be introduced by the manipulation of the bait region, recombinant alpha 2M (r alpha 2M) was produced in which the primary cleavage site was replaced by a heptapeptide containing the cleavage specificity of tobacco etch virus (TEV) protease. This protease is not inhibited by wild-type alpha 2M. The r alpha 2M, produced in an episomal expression system, was fully functional and able to inhibit the tobacco etch virus protease according to its normal ‘trap’ mechanism. The manipulation of the bait region of alpha 2M thus allows the design of new, specific protease inhibitors.

scholarly journals Directed evolution improves the catalytic efficiency of TEV protease

2019 ◽  
Author(s):  
Mateo I Sanchez ◽  
Alice Y Ting
Keyword(s):  
Directed Evolution ◽  
Catalytic Properties ◽  
Light Exposure ◽  
Catalytic Efficiency ◽  
Tobacco Etch Virus ◽  
Sequence Specificity ◽  
Tobacco Etch Virus Protease ◽  
Tev Protease ◽  
Wide Range ◽  
Catalytic Rate

AbstractTobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV’s cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications.

scholarly journals Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

2021 ◽  
pp. 100879
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Khang Diep ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Bait Region ◽  
Α2 Macroglobulin

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 Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition

2015 ◽  
Vol 112 (27) ◽  
pp. 8290-8295 ◽  
Author(s):  
Irene Garcia-Ferrer ◽  
Pedro Arêde ◽  
Josué Gómez-Blanco ◽  
Daniel Luque ◽  
Stephane Duquerroy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Large Cell ◽  
Structural Rearrangement ◽  
Gram Negative Bacteria ◽  
Human Gut ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Thioester Bond ◽  
Peptidase Inhibitor

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric “snap trap.”

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