title: Expression of G protein-coupled receptors in Mammalian cells.
abstract: G protein-coupled receptors (GPCRs) are cell surface proteins and play crucial role in mediating effective communication between extracellular and intracellular milieu of the cell. To understand the structure and function of these membrane proteins, it is imperative to express the proteins in a functional form and in sufficient quantities. However, heterologous expression of GPCRs in sufficient amounts for structural studies is a daunting task and over the years researchers have tried various expression systems to achieve this goal. Here, we describe a method developed in the laboratory of Dr. Har Gobind Khorana and successfully used over the past decade to express GPCRs and other membrane proteins in mammalian cells. Improvements to this method include use of genes codon optimized for expression in mammalian cells, use of expression tags at N-terminus, and affinity tags at the N-terminus and C-terminus of GPCRs. It also provides details for the process of selection for stably transfected clones, and to identify clones that are effectively expressing the GPCR of interest on the cell surface using flow cytometry. Purification of the overexpressed GPCR involves use of affinity chromatography. The method discussed here can also be effectively used to express cytotoxic proteins such as some constitutively active GPCRs. The method is robust and reliable to express GPCRs.
authors: Department of Oral Biology, College of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Raja
authors: R
authors: Chakraborty
authors: Department of Oral Biology, College of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Bing
authors: B
authors: Xu
authors: Department of Oral Biology, College of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Rajinder P
authors: RP
authors: Bhullar
authors: Department of Oral Biology, College of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address: prashen.chelikani@umanitoba.ca.
authors: Prashen
authors: P
authors: Chelikani
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D015870:Gene Expression; D005813:Genes, Synthetic; D057809:HEK293 Cells; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods
chemical_list: D003062:Codon; D043562:Receptors, G-Protein-Coupled
keywords: Codon-optimized genes; Constitutively active GPCRs; Expression studies; G protein-coupled receptors; Mammalian cell lines