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.

Figure 1

Flow chart of the overexpression of membrane proteins in HEK293S GnTI⁻ cells. After one or more rounds of screening, a few potential candidates are chosen for large-scale protein expression.

Figure 2

Map of BacMam Expression Vector. For expression in mammalian cells, genes of interest are cloned into the multiple cloning site behind the CMV promoter using unique restriction sites. Elements that are important for high level expression are shown, including those that are important for transcription initiation (CMV promoter), transcription termination (SV40 poly A late signal), mRNA processing (intron and WPRE motif). Also indicated are the elements important for insect cell expression and baculovirus amplification including promoters (polh and p10), terminators (SV40 and HSVtk), transposon elements (Tn7L and Tn7R), and resistance markers (ampicillin and gentamicin).

Figure 3

Screening constructs by small scale transient transfection in HEK293S GnTI⁻ cells and FSEC. 1 μg of N-terminal eGFP tagged full length cASIC1 or cASIC463 (cASIC1 truncated 64 residues from the carboxy termini) subcloned into pEG BacMam was transfected separately into HEK293S GnTI⁻ cells, as described in the text. Cells were harvested 48 h after transfection and solubilized extracts were analyzed by FSEC to determine the behavior of the fusion proteins. c.p.s., counts per second.

Figure 4

Time course of expression of cASIC and GluCl in HEK293S GnTI⁻ cells. HEK293S GnTI⁻ cells (3×10⁶ cells/ml) were transduced with BacMam virus for cASIC463 (a and b) or GluCl-EGFP (c and d) at a MOI of 1 and incubated at 37 °C. After 8 h, 10 mM sodium butyrate was added and cultures were left at 37 °C or shifted to 30 °C. Time points were taken at the indicated times, samples were frozen and later solubilized with 40 mM C₁₂M in TBS pH 8 and analyzed by FSEC. (a and c) Representative FSEC profiles from cASIC463 (a) and GluCl-EGFP (c) detected by GFP fluorescence. (b and d) Comparison of expression at 37 °C and 30 °C from cASIC463 and (b) GluCl-EGFP (d) based on the peak area estimated by Gaussian fitting. (e) Western blot analysis using an anti-GFP polyclonal antibody of peak fractions isolated from FSEC profiles from (a) cASIC463 and (c) GluCl-EGFP.

Figure 5

Effect of histone deacetylase inhibitors on the expression of cASIC and GluCl in HEK293S GnTI⁻ cells. (a and b) Comparison of cASIC expression with and without sodium butyrate. Representative FSEC profiles from cASIC463 detected by GFP fluorescence (a) and comparison of expression from cASIC463 based on the peak area estimated by Gaussian fitting (b) in the presence or absence of sodium butyrate. (c) Representative FSEC profiles from GluCl-EGFP in the presence of sodium butyrate or valproic acid.

Figure 6

Comparison of expression time course in HEK293S GnTI⁻ cells and Sf9 cells. HEK293S GnTI⁻ cells were transduced with BacMam virus for cASIC463 (a and b) or GluCl-EGFP (c and d) as described above. Sf9 cells (3×10⁶ cells/ml) were infected with baculovirus cASIC463 (a and b) or GluCl-EGFP (c and d) and placed at 27 °C. After 18 h, Sf9 cultures were left at 27 °C or shifted to 20 °C. Time points were taken, frozen and solubilized as described above. (a and c) Representative FSEC profiles from cASIC463 (a) and GluCl-EGFP (c) detected by GFP fluorescence. (b and d) Comparison of expression from cASIC463 (b) GluCl-EGFP (d) based on the peak area estimated by Gaussian fitting.