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

Anal Biochem. 2009 Mar 15;386(2):147-55. doi: 10.1016/j.ab.2008.12.016. Epub 2008 Dec 24.

Abstract

G-protein-coupled receptors (GPCRs) represent approximately 3% of human proteome and the most prominent class of pharmacological targets. Despite their important role in many functions, only the X-ray structures of rhodopsin, and more recently of the beta(1)- and beta(2)-adrenergic receptors, have been resolved. Structural studies of GPCRs require that several tedious preliminary steps be fulfilled before setting up the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. Here we report on screening expression conditions of approximately 100 GPCRs in Escherichia coli with a view to obtain large amounts of inclusion bodies, a prerequisite to the subsequent refolding step. A set of optimal conditions, including appropriate vectors (Gateway pDEST17oi), strain (C43), and fermentation at high optical density, define the best first instance choice. Beyond this minimal setting, however, the rate of success increases significantly with the number of conditions tested. In contrast with experiments based on a single GPCR expression, our approach provides statistically significant results and indicates that up to 40% of GPCRs can be expressed as inclusion bodies in quantities sufficient for subsequent refolding, solubilization, and purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Humans
  • Inclusion Bodies / metabolism*
  • Mammals
  • Protein Engineering / methods
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / isolation & purification
  • Receptors, G-Protein-Coupled / metabolism*

Substances

  • Receptors, G-Protein-Coupled