title: Rapid and facile recombinant expression of bovine rhodopsin in HEK293S GnTI(-) cells using a PiggyBac inducible system.
abstract: Rhodopsin is a class A G protein-coupled receptor (GPCR) that provides important insights into the structure and function of the GPCR superfamily. Bovine rhodopsin is widely used as a model for GPCRs and was the first GPCR whose X-ray crystal structure was solved. One of the advantages of rhodopsin is that it is abundant in native tissue, and as a result, milligram quantities can be purified from the retinal rod cells of bovine eyes. Nonetheless, the study of GPCR conformation and dynamics, e.g., by electron paramagnetic resonance or (19)F nuclear magnetic resonance spectroscopy, typically requires mutagenesis to enable site-directed labeling of the protein. Mutations are also of great importance as they can stabilize the receptor and can be necessary to study different receptor conformations. Recombinant production of rhodopsins for biophysical studies has been achieved in different systems, including mammalian, insect, and yeast cells in culture, and from Drosophila melanogaster and Caenorhabditis elegans tissue. The piggyBac (PB) transposon system is used for gene delivery into a variety of cell types (e.g., HEK293 and CHO cells, fibroblasts, stem cells) and living organisms (e.g., honeybees, pigs, chicken, mice). Recently, the PB transposon has been described as an efficient tool for inducible protein expression in HEK293T and HEK293S N-acetylglucosaminyltransferase I-deficient (GnTI(-)) cells. This chapter describes a protocol for using the PB-based system for inducible expression of bovine rhodopsin in HEK293S GnTI(-) cells. Using this protocol, we expressed and purified 26 rhodopsin mutants to be used for site-directed spin labeling.
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors: Lydia N
authors: LN
authors: Caro
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors: Zhijie
authors: Z
authors: Li
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors: Aidin R
authors: AR
authors: Balo
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors: Ned
authors: N
authors: Van Eps
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Electronic address: james.rini@utoronto.ca.
authors: James M
authors: JM
authors: Rini
authors: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Electronic address: oliver.ernst@utoronto.ca.
authors: Oliver P
authors: OP
authors: Ernst
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000235:genetics; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012243:Rhodopsin / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods
chemical_list: D004251:DNA Transposable Elements; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012243:Rhodopsin; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
keywords: HEK293S GnTI(−); Recombinant mammalian expression; Rhodopsin mutants; Site-directed spin labeling (SDSL)