ArticleProceedings of the National Academy of Sciences of the United States of America2013
Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 57 papers.
What it found
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Who cites it
57 citing papers in PubMed, 138 citations in OpenAlex.
- Noise-Guided Design of Synthetic Protein Waves in Living Cells.ACS synthetic biology · 2026Article
- Decoding the substrate specificity landscape of a promiscuous enzyme through multi-substrate mutational scanning.Nature communications · 2026Article
- A gasdermin-based life-death evolution system for reprogramming protease specificity.Nature chemical biology · 2026Article
- High-Throughput Activity Reprogramming of Proteases (HARP).ACS chemical biology · 2025Article
- Engineering the Tobacco Etch Virus Protease toward a Platform for Traceless Cleavage Using Distal Site Prediction and Smart Library Design.ACS synthetic biology · 2025Article
- Protease engineering: Approaches, tools, and emerging trends.Biotechnology advances · 2025Review
- Data-driven protease engineering by DNA-recording and epistasis-aware machine learning.Nature communications · 2025Article
- PERRC: Protease Engineering with Reactant Residence Time Control.ACS synthetic biology · 2025Article
- Enzyme-substrate co-display on yeast empowers engineering of tobacco etch virus protease activity.Protein engineering, design & selection : PEDS · 2025Article
- Article
- High-Resolution Substrate Specificity Profiling of SARS-CoV-2 MACS chemical biology · 2024Article
- An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity.Cell chemical biology · 2024Article
- Synthetic Biology Meets CaCells · 2024Review
- Leveraging yeast sequestration to study and engineer posttranslational modification enzymes.Biotechnology and bioengineering · 2024Review
- Mechanism of Mutation-Induced Effects on the Catalytic Function of TEV Protease: A Molecular Dynamics Study.Molecules (Basel, Switzerland) · 2024Article
- Modular and integrative activity reporters enhance biochemical studies in the yeast ER.Protein engineering, design & selection : PEDS · 2024Article
- Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display.Journal of the American Chemical Society · 2023Article
- Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.Microbial cell factories · 2023Article
- Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture.Nature communications · 2023Article
- Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS.Methods in molecular biology (Clifton, N.J.) · 2023Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 2 countries.
Funding
Abstract
Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ↓S substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu- and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.