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

April Goehring, Chia Hsueh Lee, Kevin H. Wang, Jennifer Carlisle Michel, Derek P. Claxton, Isabelle Baconguis, Thorsten Althoff, Suzanne Fischer, K. Christopher Garcia, Eric Gouaux

Research output: Contribution to journalArticle

118 Citations (Scopus)

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-His 8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI â ' (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.

Original languageEnglish (US)
Pages (from-to)2574-2585
Number of pages12
JournalNature Protocols
Volume9
Issue number11
DOIs
StatePublished - Nov 27 2014

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Baculoviridae
Screening
Membrane Proteins
Cells
Acid Sensing Ion Channels
Proteins
Size exclusion chromatography
X ray crystallography
X Ray Crystallography
Caenorhabditis elegans
Gel Chromatography
Transfection
Chickens
Assays
Suspensions
Plasmids
Fluorescence
Molecules
Experiments

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. / Goehring, April; Lee, Chia Hsueh; Wang, Kevin H.; Michel, Jennifer Carlisle; Claxton, Derek P.; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K. Christopher; Gouaux, Eric.

In: Nature Protocols, Vol. 9, No. 11, 27.11.2014, p. 2574-2585.

Research output: Contribution to journalArticle

Goehring, A, Lee, CH, Wang, KH, Michel, JC, Claxton, DP, Baconguis, I, Althoff, T, Fischer, S, Garcia, KC & Gouaux, E 2014, 'Screening and large-scale expression of membrane proteins in mammalian cells for structural studies', Nature Protocols, vol. 9, no. 11, pp. 2574-2585. https://doi.org/10.1038/nprot.2014.173
Goehring, April ; Lee, Chia Hsueh ; Wang, Kevin H. ; Michel, Jennifer Carlisle ; Claxton, Derek P. ; Baconguis, Isabelle ; Althoff, Thorsten ; Fischer, Suzanne ; Garcia, K. Christopher ; Gouaux, Eric. / Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. In: Nature Protocols. 2014 ; Vol. 9, No. 11. pp. 2574-2585.
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