Refolding aggregation-prone GPCRs and G-protein α subunits from inclusion bodies expressed in Escherichia coli

BIOT 483

Emily C. McCusker, mccusker@udel.edu1, Steven E. Bane2, and Anne Skaja Robinson, asr@udel.edu2. (1) Department of Chemistry and Biochemistry, University of Delaware, 150 Academy Street, Newark, DE 19711, (2) Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, DE 19711
G protein-coupled receptors (GPCRs) are seven-helix transmembrane proteins involved in numerous cellular process and diseases in humans; they are the targets of over 30% of drug discovery efforts. Heterotrimeric G-proteins are membrane-associated signaling partners of GPCRs that initiate the signaling cascades, and therefore both GPCR and G-proteins are of great interest for biophysical characterization and high-resolution structural studies, necessitating their large-scale production. We have expressed the neurokinin NK1R, a class A (rhodopsin-like) GPCR, and representative members of the Gα family as inclusion bodies in Escherichia coli and determined best-case refolding strategies to minimize aggregation. We found that NK1R expression activated the stress response of E. coli, but that NK1R was not degraded, unlike many other GPCR that were expressed as inclusion bodies. We have purified milligram amounts of inactive NK1R from detergent-solubilized inclusion bodies for use in characterization and refolding studies. We have also developed a novel strategy for refolding Gα subunits expressed as E. coli inclusion bodies. The refolded Gα subunits are monomeric, bind GDP, and have native-like α-helicity.
 

Biophysical and Biomolecular Symposium: Protein Aggregation
2:00 PM-5:25 PM, Thursday, August 23, 2007 BCEC -- 107C, Oral

Division of Biochemical Technology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007