Efficient Expression and Immunoaffinity Purification of Human Trace Amine-Associated Receptor 5 from E. coli Cell-Free System

Xiaoqiang      Wang; Ying      Cui; Jiqian      Wang

doi:10.2174/0929866511320040012

Abstract

G protein-coupled receptors (GPCRs) represent attractive targets for bioactive and drug discovery programs. The availability of purified receptors in milligram quantities is essential to spur the advancement of protein-based analyses in these programs, although it is still a challenging goal to achieve. Here we report the production of a bioengineered GPCR of human trace amine-associated receptor 5 (hTAAR5) from an E. coli cell-free system. Both the hTAAR5 and hTAAR5-T4 lysozyme fusion proteins (hTAAR5-T4L) were cloned and expressed in this process, with the latter designed for further protein crystallization trials. The detergent Brij-35 was found to solubilize the produced hTAAR5 and hTAAR5-T4L effectively. Immunoaffinity purification in combination with gel filtration was employed to purify the receptors to high homogeneity. The final yields of monomeric hTAAR5 and hTAAR5-T4L from a 1 mL cell-free reaction were 0.4 mg and 0.5 mg, respectively. Circular Dichroism (CD) spectroscopy indicated that both hTAAR5 and hTAAR5- T4L were correctly folded after purification, with characteristic high -helical contents (>45%).

Keywords: G protein-coupled receptor, human trace amine-associated receptor 5, efficient expression, immunoaffinity purification, cell-free system, detergent, drug discovery, crystallization, Immunoaffinity, gel filtration