skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein

Journal Article · · Protein Expression and Purification
 [1];  [2];  [3];  [1];  [3];  [3];  [1];  [3]; ORCiD logo [1]
  1. University of North Carolina, Chapel Hill, NC (United States)
  2. Univ. of Wisconsin, Madison, WI (United States); National Institute of Technology, Fukuoka (Japan). Kitakyushu College
  3. Univ. of Wisconsin, Madison, WI (United States)
+ Show Author Affiliations

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.

Research Organization:
University of North Carolina, Chapel Hill, NC (United States)
Sponsoring Organization:
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)
Grant/Contract Number:
FG02-05ER15671; R01GM065989; MCB-0718202; U54GM094584; FG02-05er15671
OSTI ID:
1534223
Alternate ID(s):
OSTI ID: 1323974
Journal Information:
Protein Expression and Purification, Vol. 126, Issue C; ISSN 1046-5928
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 8 works
Citation information provided by
Web of Science

References (29)

Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system journal October 2000
The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex journal May 2009
Cell-Free Protein Synthesis for Functional and Structural Studies book October 2013
The Plant Heterotrimeric G-Protein Complex journal June 2007
GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling journal October 2007
Practical cell-free protein synthesis system using purified wheat embryos journal January 2010
Cell-free protein synthesis: Applications come of age journal September 2012
Direct Measurement of Thermal Stability of Expressed CCR5 and Stabilization by Small Molecule Ligands journal February 2011
The nanodisc: a novel tool for membrane protein studies journal August 2009
Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins journal August 2002
Eukaryotic G Protein Signaling Evolved to Require G Protein-Coupled Receptors for Activation journal May 2013
Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses journal October 2008
Heterotrimeric G Protein–Coupled Signaling in Plants journal April 2014
Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis journal September 2012
Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform: Biotechnol. Prog. journal March 2013
Rapid Incorporation of Functional Rhodopsin into Nanoscale Apolipoprotein Bound Bilayer (NABB) Particles journal April 2008
Dissociation of Heterotrimeric G Proteins in Cells journal June 2008
Monomeric Rhodopsin Is the Minimal Functional Unit Required for Arrestin Binding journal June 2010
A Seven-Transmembrane RGS Protein That Modulates Plant Cell Proliferation journal September 2003
A Spatial Focusing Model for G Protein Signals journal February 2003
Human protein factory for converting the transcriptome into an in vitro–expressed proteome journal November 2008
The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes journal December 2014
Functional reconstitution of β 2 -adrenergic receptors utilizing self-assembling Nanodisc technology journal May 2006
G Protein Activation without a GEF in the Plant Kingdom journal June 2012
A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein journal April 2007
Directed Self-Assembly of Monodisperse Phospholipid Bilayer Nanodiscs with Controlled Size journal March 2004
Efficient Coupling of Transducin to Monomeric Rhodopsin in a Phospholipid Bilayer journal February 2008
Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding journal October 2010
Transducin Activation by Nanoscale Lipid Bilayers Containing One and Two Rhodopsins journal March 2007

Similar Records

Related Subjects

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