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Nat Protoc. 2020 Jan;15(1):144-160. doi: 10.1038/s41596-019-0259-y. Epub 2019 Nov 29.

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

Author information

1
Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, USA. martin.audet2@usherbrooke.ca.
2
Department of Pharmacology and Physiology, Pharmacology Institute of Sherbrooke, CHUS Research Center, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada. martin.audet2@usherbrooke.ca.
3
Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, USA.
4
Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, USA. stevens@usc.edu.

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.

PMID:
31784719
DOI:
10.1038/s41596-019-0259-y

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