Small-scale approach for precrystallization screening in GPCR X-ray crystallography
| Autor: | Jeffrey Velasquez, Raymond C. Stevens, Kelly Villers, Meihua Chu, Chris Hanson, Martin Audet |
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| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
Chemistry Uv absorbance Sf9 General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 0302 clinical medicine Protein sequencing Cell culture Yield (chemistry) Protein purification Biophysics Receptor 030217 neurology & neurosurgery 030304 developmental biology G protein-coupled receptor |
| Zdroj: | Nature Protocols. 15:144-160 |
| ISSN: | 1750-2799 1754-2189 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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