title: Efficient expression and immunoaffinity purification of human trace amine-associated receptor 5 from E. coli cell-free system.
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 α-helical contents ( > 45%).
authors.affiliation: State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266580, P.R. China. wangxq001@upc.edu.cn
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Cui
authors.forename: Jiqian
authors.initials: J
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002474:Cell-Free System; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000095383:Trace Amine-Associated Receptors
chemical_list: D003902:Detergents; D009113:Muramidase; D011092:Polyethylene Glycols; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C583307:TAAR5 protein, human; D000095383:Trace Amine-Associated Receptors; C515901:Brij 35