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Small-scale approach for precrystallization screening in GPCR X-ray crystallography

Nature Protocols volume 15, pages 144–160 (2020)Cite this article

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Abstract

G protein–coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor’s protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

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Fig. 1: Overview of the procedure.
Fig. 2: GPCR expression in small-scale (4-ml) Sf9 cell culture.
Fig. 3: Size-exclusion chromatography of purified small-scale culture expressing different EP3 receptor constructs.

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Data availability

The datasets generated and analyzed here are available from the authors upon request.

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Acknowledgements

This research was supported by NIH/NIGMS Protein Structure Initiative U54GM094618 (R.C.S.). M.A. was supported by a Canadian Institute of Health and Research (CIHR) Postdoctoral Fellowship Award. We thank A. Walker for assistance in manuscript preparation and F. Badeaux and E. Audet-Badeaux for their support.

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Author notes

  1. Martin Audet

    Present address: Department of Pharmacology and Physiology, Pharmacology Institute of Sherbrooke, CHUS Research Center, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada

Authors and Affiliations

  1. Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, USA

    Martin Audet, Kelly Villers, Jeffrey Velasquez, Meihua Chu, Chris Hanson & Raymond C. Stevens

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  1. Martin Audet
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Contributions

M.A. designed the study; cloned the constructs; and performed the baculovirus production, receptor expression, and receptor purification and characterization. J.V. cloned the constructs; K.V., M.C, and C.H. performed baculovirus production and receptor expression; M.A. and R.C.S. supervised the project.

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Correspondence to Martin Audet or Raymond C. Stevens.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Gopala Krishna Aradhyam and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Audet, M. et al. Nat. Chem. Biol. 15, 11–17 (2019): https://www.nature.com/articles/s41589-018-0160-y

Integrated supplementary information

Supplementary Fig. 1 Illustration of the gating strategy used in the flow cytometry experiment.

a, Plot of front scatter (FSC) against side scatter (SSC) signals shows that Sf9 insect cells growth as a homogeneous and monodispersed suspension culture. For GP64-PE and FLAG-FITC experiments, we measured 2000 events that show an FSC signal above 180 counts. We are not gating SSC signal. b, Typical GP64-PE experiment to monitor baculovirus budding on the surface of Sf9 cells after bacmid transfection. Positive GP64-PE signal is set above 20 counts. Non-transfected Sf9 cells are used as a negative control and shown in blue, Sf9 cells transfected with a bacmid containing the coding sequence of A2A-BRIL is shown in magenta. The gate was set at 20 counts for positive GP64-PE signal. c-d, Typical FLAG-FITC experiment to monitor c, cell surface and d, total receptor expression in Sf9 cells after baculovirus infection. Non-infected Sf9 cells are used as negative control and shown in blue. Sf9 cell expressing A2A-BRIL receptor fusion is shown in magenta. The gate was set at 30 counts for positive FLAG-FITC signal for both c, cell surface and d, total receptor expression. The gates are depicted on the graphs as solid lines.

Supplementary Fig. 2 Illustration of the EP3-rub sequence and truncations.

a, n- and b, c-terminal truncations of EP3-rub reference construct. The non-truncated EP3-rub is the isoform D of EP3 receptor fused with a rubredoxin flanked by a Gly-Ser linker on both sides (EP3-rub). The rubredoxin is inserted in the third intracellular loop of EP3 by replacing the receptor residues 260-267. A FLAG-tag and a 10 x his-tag were fused at both n- and c-terminus respectively. EP3 receptor residue number are indicated above each cartoon.

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Supplementary Information

Supplementary Figures 1 and 2 and Supplementary Table 1

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Audet, M., Villers, K., Velasquez, J. et al. Small-scale approach for precrystallization screening in GPCR X-ray crystallography. Nat Protoc 15, 144–160 (2020). https://doi.org/10.1038/s41596-019-0259-y

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