title: Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.
abstract: Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.
authors.affiliation: 1] Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: April
authors.initials: A
authors.lastname: Goehring
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Chia-Hsueh
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Kevin H
authors.initials: KH
authors.lastname: Wang
authors.affiliation: 1] Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Jennifer Carlisle
authors.initials: JC
authors.lastname: Michel
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Derek P
authors.initials: DP
authors.lastname: Claxton
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Baconguis
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Althoff
authors.affiliation: Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Fischer
authors.affiliation: 1] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [2] Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
authors.affiliation: 1] Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Gouaux
mesh_terms: D062565:Acid Sensing Ion Channels / Q000235:genetics / Q000378:metabolism; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics; D006801:Humans; D008322:Mammals; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D062565:Acid Sensing Ion Channels; D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D008565:Membrane Proteins; D011994:Recombinant Proteins; C109777:glutamate-gated chloride channels; D049452:Green Fluorescent Proteins; D006639:Histidine; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I