Refolding of G-protein-coupled receptors from inclusion bodies produced in Escherichia coli

1999 ◽  
Vol 27 (6) ◽  
pp. 908-912 ◽  
Author(s):  
Hans Kiefer ◽  
Klaus Maier ◽  
Reiner Vogel
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
Inclusion Bodies ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli

2012 ◽  
Vol 14 (5) ◽  
pp. 591-602 ◽  
Author(s):  
Georgios Skretas ◽  
Tomohiro Makino ◽  
Navin Varadarajan ◽  
Mark Pogson ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

Refolding of G protein-coupled receptors from inclusion bodies produced in E. coli

1999 ◽  
Vol 27 (5) ◽  
pp. A141-A141
Author(s):  
H. Kiefer ◽  
K. Maier ◽  
R. Vogel
Keyword(s):  
G Protein ◽  
Inclusion Bodies ◽  
G Protein Coupled Receptors ◽  
E Coli ◽  
G Protein Coupled

scholarly journals Multi-Copy Genes that Enhance the Yield of Mammalian G Protein-Coupled Receptors in Escherichia coli

2012 ◽  
Vol 29 ◽  
pp. S146
Author(s):  
Georgios Skretas ◽  
Tomohiro Makino ◽  
Navin Varadarajan ◽  
Mark Pogson ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247689
Author(s):  
Bastian Heim ◽  
René Handrick ◽  
Marcus D. Hartmann ◽  
Hans Kiefer
Keyword(s):  
G Protein ◽  
Inclusion Bodies ◽  
Protein Unfolding ◽  
G Protein Coupled Receptors ◽  
Orphan Receptor ◽  
Cubic Phase ◽  
E Coli ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.

scholarly journals Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli

2010 ◽  
Vol 76 (17) ◽  
pp. 5852-5859 ◽  
Author(s):  
Georgios Skretas ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
G Protein ◽  
Protein Production ◽  
Genetic Selection ◽  
G Protein Coupled Receptors ◽  
Efficient Production ◽  
E Coli ◽  
G Protein Coupled

ABSTRACT The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme β-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.

Expression of G-protein coupled receptors in Escherichia coli for structural studies

2010 ◽  
Vol 75 (7) ◽  
pp. 881-891 ◽  
Author(s):  
L. E. Petrovskaya ◽  
A. A. Shulga ◽  
O. V. Bocharova ◽  
Ya. S. Ermolyuk ◽  
E. A. Kryukova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Structural Studies ◽  
G Protein Coupled

Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies

2009 ◽  
Vol 386 (2) ◽  
pp. 147-155 ◽  
Author(s):  
Kerstin Michalke ◽  
Marie-Eve Gravière ◽  
Céline Huyghe ◽  
Renaud Vincentelli ◽  
Renaud Wagner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
G Protein ◽  
High Throughput ◽  
Inclusion Bodies ◽  
Large Scale ◽  
Receptor Expression ◽  
Scale Production ◽  
Expression In Escherichia Coli ◽  
Large Scale Production ◽  
G Protein Coupled
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