Schlesinger Ramona, Cousin Anneliese, Granzin Joachim, Batra-Safferling Renu
Experimental Physics: Genetic Biophysics, Freie Universität Berlin, Berlin, Germany.
Institute of Complex Systems (ICS-6), Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany.
Methods Cell Biol. 2017;142:159-172. doi: 10.1016/bs.mcb.2017.07.003. Epub 2017 Sep 4.
Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column. Highly purified arrestins can be concentrated up to 15mg/mL using ultrafiltration and can be stored in the frozen state for several months without any loss of functionality.
蛋白质纯度和产量是使用X射线晶体学、核磁共振及其他几种生物物理方法等结构技术成功进行蛋白质表征的两个关键参数。酿酒酵母是用于重组蛋白过表达及后续纯化的常用真核模式系统之一。在此，我们描述了一种从酵母中克隆、过表达、纯化和结晶抑制蛋白-1及其剪接变体p44的方案。纯化方案包括在链霉亲和素柱上进行单亲和层析步骤。使用超滤可将高度纯化的抑制蛋白浓缩至15mg/mL，并且可以冷冻状态保存数月而不会有任何功能丧失。
