Tobacco Etch Virus protease: A shortcut across biotechnologies

Cesaratto, Francesca; Burrone, Óscar R.; Petris, Gianluca

doi:10.1016/j.jbiotec.2016.06.012

Cited by 50 publications

(46 citation statements)

References 108 publications

(174 reference statements)

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“…1a). As 2A allows co-translation of separate proteins encoded in a single open reading frame (ORF), it has been largely used for several biotechnological applications [27][28][29][30]. Mutation of the last Pro into a STOPcodon was reported to cause stalling of translation in in vitro systems (wheat germ and rabbit reticulocyte lysates) [31,32], making it an interesting model to study ribosome stalling in living human cells.…”

Section: Introductionmentioning

confidence: 99%

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon

Cesaratto

Sasset

Myers

et al. 2019

Journal of Molecular Biology

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Translational stalling of ribosome bound to endoplasmic reticulum (ER) membrane requires an accurate clearance of the associated polypeptides, which is not completely understood in mammals. We characterized in mammalian cells the model of ribosomal stalling at the STOP-codon based on proteins tagged at the C-terminus with the picornavirus 2A peptide followed by a termination codon instead of the Proline (2A*). We exploited the 2A* stalling model to characterize the pathway of degradation of ER-targeted polypeptides. We report that the ER chaperone BiP/GRP78 is a new main factor involved. Moreover, degradation of the ER-stalled polypeptides required the activities of the AAA-ATPase VCP/p97, its associated deubiquitinylase YOD1, the ribosome-associated ubiquitin ligase Listerin and the proteasome. In human proteome, we found two human C-terminal amino acid sequences that cause similar stalling at the STOP-codon. Our data suggest that translational stalling at the ER membrane activates protein degradation at the interface of ribosomal-and ERassociated quality control systems.

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Section: Introductionmentioning

confidence: 99%

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon

Cesaratto

Sasset

Myers

et al. 2019

Journal of Molecular Biology

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“…This greatly increases the chances of identifying ligands with highly selective binding properties. We chose to validate the approach using Tobacco Etch Virus (TEV) protease since it is known to have well-defined primary sequence selectivity yet there are currently no reported potent inhibitors of this target [26]. Our screening efforts using a cyclization linker containing a vinyl sulfone warhead identified a series of peptide sequences that irreversibly inhibited TEV only when cyclized.…”

Section: Introductionmentioning

confidence: 99%

A phage display approach to identify highly selective covalent binders

Chen

Bogyo

2019

Preprint

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Molecules that bind macromolecular targets through direct covalent modification have found widespread applications as activity-based probes (ABPs) and as irreversible drugs. Covalent binders can be used to dynamically monitor the activity of enzymes in complex cellular environments, identify targets and induce permanent binding/inhibition of therapeutically important biomolecules. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, robust and unbiased screening method to identify new classes of covalent binding ligands from highly diverse pools of candidate molecules. Here we describe the development of a phage display method to screen for highly selective covalent binding ligands. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic 'warhead' to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that selectively and irreversibly inhibited a cysteine protease with nanomolar potency, exceptional specificity and increased serum stability compared to a linear peptide containing the same electrophile. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent binding ligands for diverse molecular targets.

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“…While there are huge benefits of tags, their incorporation on a protein of interest can hinder the activity of the tagged protein, therefore, it is generally advised to remove the tags. The main advantage of TEV protease compared to other proteases, such as Factor Xa, and thrombin, is that it is extremely selective for its recognition site, thus its accuracy ensures the protein is not cleaved incorrectly, inactivating the protein …”

Section: Introductionmentioning

confidence: 99%

“…A major use of tobacco etch virus (TEV) protease in biotechnology is for the cleavage of affinity tags used for purification of recombinantly expressed proteins. 1 Tags are fused with proteins of interest, generally to assist with purification, but they can also act to increase yields, protect them from intracellular proteolysis and in the case of the maltose binding protein, aid in solubility. 2 While there are huge benefits of tags, their incorporation on a protein of interest can hinder the activity of the tagged protein, therefore, it is generally advised to remove the tags.…”

Section: Introductionmentioning

confidence: 99%

“…The main advantage of TEV protease compared to other proteases, such as Factor Xa, and thrombin, is that it is extremely selective for its recognition site, thus its accuracy ensures the protein is not cleaved incorrectly, inactivating the protein. 1 TEV protease (EC number 3.4.22.44) recognizes the linear epitope sequence E-Xaa-Xaa-Y-X aa -Q-(G/S), with cleavage occurring between the Q and the G/S amino acid residues. The optimal recognition site of TEV protease was shown to be ENLYFQG, 3 which is the sequence present in all of the cleavable poly-histidine tagged (his-tagged) proteins used in this research.…”

Section: Introductionmentioning

confidence: 99%

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Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold

Raynes

Domigan

Pearce

et al. 2018

Biotechnology Progress

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Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization. Immobilization of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilized active TEV protease. The bead retained activity over several uses and successfully cleaved a poly-histidine tag from several his-tagged proteins. This is first time that TEV protease has been immobilized to insulin amyloid fibrils, or any protein based support. Such functionalized surface assembled amyloid fibrils show promise as a novel nanosupport for the creation of functional bionanomaterials, for example, active surface coatings for the production of fine chemicals, chemical detoxification, or biosensing. Insulin amyloid fibrils provide a new nanosupport for the immobilization of TEV protease, which could allow for the reuse of the enzyme, saving on production costs for recombinantly expressed poly-histidine tagged proteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

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Tobacco Etch Virus protease: A shortcut across biotechnologies

Cited by 50 publications

References 108 publications

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon

A phage display approach to identify highly selective covalent binders

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold

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