New method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside the host cell

MBio
Gregory M WierJon P Boyle

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRS(NLL)) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed "click" reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRS(NLL)-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRS(NLL)-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRS(NLL)-expressin...Continue Reading

References

Oct 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·M F Cesbron-DelauwJ P Lecocq
Nov 1, 1994·Experimental Parasitology·E R Pfefferkorn, S E Borotz
Mar 6, 1998·Molecular and Cellular Biology·H J DrabkinU L Rajbhandary
Nov 11, 1999·Proceedings of the National Academy of Sciences of the United States of America·E ZutherP Gornicki
Mar 29, 2000·Biopolymers·P J Beuning, K Musier-Forsyth
Feb 28, 2001·Proceedings of the National Academy of Sciences of the United States of America·J JelenskaP Gornicki
Dec 10, 2002·Current Opinion in Chemical Biology·Takahiro Hohsaka, Masahiko Sisido
May 5, 2004·Chemistry & Biology·Anna E Speers, Benjamin F Cravatt
Jun 15, 2004·Lancet·J G Montoya, O Liesenfeld
Jun 14, 2006·Proceedings of the National Academy of Sciences of the United States of America·Daniela C DieterichErin M Schuman
Jun 28, 2006·Proceedings of the National Academy of Sciences of the United States of America·A James LinkDavid A Tirrell
Dec 22, 2007·Chembiochem : a European Journal of Chemical Biology·Aijun WangKenneth Grabstein
Sep 2, 2008·Proceedings of the National Academy of Sciences of the United States of America·Giel G van DoorenBoris Striepen
Sep 9, 2008·Bioorganic & Medicinal Chemistry Letters·Kimberly E Beatty, David A Tirrell
Aug 12, 2009·Nature Chemical Biology·John T NgoDavid A Tirrell
Aug 27, 2009·Proceedings of the National Academy of Sciences of the United States of America·I Caglar TanrikuluDavid A Tirrell
Jun 15, 2010·Nature Neuroscience·Daniela C DieterichErin M Schuman
May 19, 2011·Proceedings of the National Academy of Sciences of the United States of America·Michael S BehnkeL David Sibley
Aug 6, 2011·Accounts of Chemical Research·John T Ngo, David A Tirrell
Oct 19, 2011·Infection and Immunity·Crystal M Tobin, Laura J Knoll
Mar 13, 2013·Proceedings of the National Academy of Sciences of the United States of America·John T NgoDavid A Tirrell
Dec 19, 2013·Proceedings of the National Academy of Sciences of the United States of America·Alborz MahdaviDavid A Tirrell
Aug 28, 2014·Connective Tissue Research·Lynn S MirigianSergey Leikin

❮ Previous
Next ❯

Citations

May 9, 2019·New Directions for Student Leadership·Adam Peck, Kathleen Callahan
May 9, 2019·New Directions for Student Leadership·Jonathan S Lewis
May 11, 2018·Proteomics·Megan H Wright
Nov 9, 2018·Frontiers in Cellular and Infection Microbiology·Magdalena FrancoSahar H El-Etr
May 30, 2019·Journal of Biological Engineering·Aya M SalehTamara L Kinzer-Ursem

❮ Previous
Next ❯

Methods Mentioned

BETA
protein folding
affinity purification
pulldowns
PCR

Software Mentioned

tRNAscan
SE

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.