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Nat Protoc. 2014 Nov;9(11):2574-85. doi: 10.1038/nprot.2014.173. Epub 2014 Oct 9.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

Author information

1
1] Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
2
Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA.
3
Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
4
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.

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.

PMID:
25299155
PMCID:
PMC4291175
DOI:
10.1038/nprot.2014.173
[Indexed for MEDLINE]
Free PMC Article

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