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

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Small-scale approach for precrystallization screening in GPCR X-ray crystallography.
Nature Protocols ( IF 13.1 ) Pub Date : 2019-11-29 , DOI: 10.1038/s41596-019-0259-y
Martin Audet _{1,

2} , Kelly Villers ₁ , Jeffrey Velasquez ₁ , Meihua Chu ₁ , Chris Hanson ₁ , Raymond C Stevens ₁

Affiliation

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.

中文翻译：

GPCR X射线晶体学中用于预结晶筛选的小规模方法。

G蛋白偶联受体（GPCR）是重要的药物靶标。了解其3D结构对于了解药物作用机制至关重要。低细胞表达，纯化产率和体外不稳定性是成功确定GPCR结构的主要障碍。需要大量的努力来优化受体的蛋白质序列和纯化程序，从而增加了预结晶过程的复杂性。在这里，我们提出了一种用于小规模预结晶筛查的程序，该程序包括通过流式细胞仪检测在草地贪夜蛾（Sf9）培养物中的GPCR表达水平，并通过尺寸排阻色谱法和UV吸光度测量评估GPCR的稳定性。该示例程序使用最小体积的Sf9细胞培养物，该培养物将产生足够量的纯化蛋白用于固有UV吸收分析，并且适合中等通量，并且具有与结晶试验相同的相同构建体和条件。该方案需要8天才能完成，并且需要细胞培养，蛋白质纯化和色谱方面的专业知识。

更新日期：2019-11-30

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