Proteases that can distinguish among different post-translational forms of tyrosine engineered using multicolor flow cytometry

Journal of the American Chemical Society
Navin VaradarajanBrent L Iverson

Abstract

We report the isolation of a highly active (k(cat)/K(M) approximately 10(5) M(-1)s(-1)) variant of the E. coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine (160-fold), sulfotyrosine (3600-fold), phosphotyrosine (>8000-fold), and phosphoserine (>8000-fold). The isolation of endopeptidase variants that can discriminate between substrates based on the post-translational modification of Tyr was made possible by implementing a multicolor flow cytometric assay for the screening of large mutant libraries. For the multicolor assay, a desired selection substrate was used simultaneously with multiple counterselection fluorescent substrates to isolate rare enzyme variants that displayed finely tuned substrate specificity. This work demonstrates that enzymes with exquisite selectivity can be isolated from large libraries using appropriate high throughput screening approaches and constitutes a critical step toward the production of a 3-nitrotyrosine-specific protease useful for proteomic applications.

References

Apr 7, 1967·Biochemical and Biophysical Research Communications·M SokolovskyB L Vallee
Nov 12, 1996·Biochemical and Biophysical Research Communications·T YamadaM Kikuchi
Apr 3, 2001·Nature Biotechnology·H ZhouR Aebersold
Sep 24, 2002·Nature Biotechnology·Stephen W SantoroPeter G Schultz
Sep 10, 2003·Protein Expression and Purification·Richard J JennyRoger L Lundblad
Aug 31, 2006·Current Opinion in Chemical Biology·Olga KhersonskyDan S Tawfik
Jun 5, 2007·Biochemistry·Matthew R ReynoldsLester I Binder
Sep 7, 2007·Journal of the American Chemical Society·Zachary A KnightKevan M Shokat
Apr 9, 2008·Nature Chemical Biology·Navin VaradarajanBrent L Iverson
Apr 25, 2008·Bioorganic & Medicinal Chemistry·Mark D Lim, Charles S Craik
May 27, 2008·Nature Biotechnology·Oliver Schilling, Christopher M Overall
Jun 24, 2008·Molecular Aspects of Medicine·Alain Doucet, Christopher M Overall
Sep 10, 2008·Angewandte Chemie·Navin VaradarajanBrent L Iverson
May 30, 2009·Nature Protocols·Navin VaradarajanBrent L Iverson

❮ Previous
Next ❯

Citations

Apr 17, 2013·Proceedings of the National Academy of Sciences of the United States of America·Li YiBrent L Iverson
Jan 16, 2013·FEBS Letters·Qing LiBrent L Iverson
May 15, 2012·Current Opinion in Structural Biology·Moshe Goldsmith, Dan S Tawfik
Apr 7, 2010·Journal of Molecular Biology·Sanjib DuttaAmy E Keating
Feb 16, 2012·Chembiochem : a European Journal of Chemical Biology·Tae Hyeon YooGeorge Georgiou
Dec 13, 2016·Proceedings of the National Academy of Sciences of the United States of America·Duc T TranBrian M Paegel
Nov 12, 2019·ACS Chemical Biology·Balakrishnan RameshNavin Varadarajan

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Proceedings of the National Academy of Sciences of the United States of America
Navin VaradarajanBrent L Iverson
Current Opinion in Biotechnology
Mark PogsonBrent L Iverson
© 2022 Meta ULC. All rights reserved