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

By A. Goehring, Chia-Hsueh Lee, Kevin H. Wang, J. C. Michel, D. P. Claxton, I. Baconguis, Thorsten Althoff, Suzanne Fischer, K. Garcia, Eric Gouaux. Nature Protocols, 2014

680 citations recorded by the source corpus.

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.

Subfields: Biomedical Engineering, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/4fd2721890f94ea34bd55cdcb9bcfbeb9054a3fa

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Citations in the corpus

• Structural basis of WLS/Evi-mediated Wnt transport and secretion (2020, background): “S1D) were both cloned into the pEG BacMam vector (Goehring et al., 2014) for baculovirus transduction of mammalian cells.”