Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein
Abstract
Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Herein, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.
- Authors:
-
[1]
- University of North Carolina, Chapel Hill, NC (United States)
- Univ. of Wisconsin, Madison, WI (United States); National Institute of Technology, Fukuoka (Japan). Kitakyushu College
- Univ. of Wisconsin, Madison, WI (United States)
- Publication Date:
- Research Org.:
- University of North Carolina, Chapel Hill, NC (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB); National Science Foundation (NSF); National Institute for General Medical Sciences (NIGMS); USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1534223
- Alternate Identifier(s):
- OSTI ID: 1323974
- Grant/Contract Number:
- FG02-05ER15671; R01GM065989; MCB-0718202; U54GM094584; FG02-05er15671
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Protein Expression and Purification
- Additional Journal Information:
- Journal Volume: 126; Journal Issue: C; Journal ID: ISSN 1046-5928
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 58 GEOSCIENCES; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology; 7-Transmembrane protein; Arabidopsis regulator of G signaling protein 1 (AtRGS1); Invitro translation; Nanodiscs; Membrane scaffold protein 1D1
Citation Formats
Li, Bo, Makino, Shin-ichi, Beebe, Emily T., Urano, Daisuke, Aceti, David J., Misenheimer, Tina M., Peters, Jonathan, Fox, Brian G., and Jones, Alan M. Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein. United States: N. p., 2016.
Web. doi:10.1016/j.pep.2016.04.016.
Li, Bo, Makino, Shin-ichi, Beebe, Emily T., Urano, Daisuke, Aceti, David J., Misenheimer, Tina M., Peters, Jonathan, Fox, Brian G., & Jones, Alan M. Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein. United States. https://doi.org/10.1016/j.pep.2016.04.016
Li, Bo, Makino, Shin-ichi, Beebe, Emily T., Urano, Daisuke, Aceti, David J., Misenheimer, Tina M., Peters, Jonathan, Fox, Brian G., and Jones, Alan M. Fri .
"Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein". United States. https://doi.org/10.1016/j.pep.2016.04.016. https://www.osti.gov/servlets/purl/1534223.
@article{osti_1534223,
title = {Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein},
author = {Li, Bo and Makino, Shin-ichi and Beebe, Emily T. and Urano, Daisuke and Aceti, David J. and Misenheimer, Tina M. and Peters, Jonathan and Fox, Brian G. and Jones, Alan M.},
abstractNote = {Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Herein, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.},
doi = {10.1016/j.pep.2016.04.016},
journal = {Protein Expression and Purification},
number = C,
volume = 126,
place = {United States},
year = {Fri May 06 00:00:00 EDT 2016},
month = {Fri May 06 00:00:00 EDT 2016}
}
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