Clark Kathleen M, Fedoriw Nadia, Robinson Katrina, Connelly Sara M, Randles Joan, Malkowski Michael G, DeTitta George T, Dumont Mark E
Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Protein Expr Purif. 2010 Jun;71(2):207-23. doi: 10.1016/j.pep.2009.12.012. Epub 2010 Jan 4.
To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents.
为了提高可用于X射线晶体学结构测定的真核跨膜蛋白（TMP）的数量和质量，我们优化了在酿酒酵母中表达的TMP的纯化方案。我们专注于一组表达量最高的内源性酵母TMP，针对这些蛋白已有成熟的生化检测方法。编码目标TMP的基因通过不依赖连接的克隆转移到一系列载体中，这些载体允许表达与C端His10和ZZ（IgG结合）结构域融合的阅读框，它们通过鼻病毒3C蛋白酶的切割位点与阅读框分开。从这些载体表达的几个TMP靶标已通过亲和色谱和凝胶过滤色谱纯化，其水平和纯度足以进行正在进行的结晶试验。最初的纯化基于在半乳糖诱导型启动子控制下的基因表达，但通过使用酵母ADH2启动子获得了更高的细胞密度和改善的表达。在纯化过程中观察到不同TMP靶标的行为存在很大差异；一些可以很容易地纯化，而另一些则不能有效地与亲和基质结合，不能有效地从基质上切割下来，或者即使在切割亲和标签后仍与基质紧密结合。使用洗涤剂浓度的比色测定法以及通过使用静态光散射、折射率和紫外吸收检测的分析尺寸排阻色谱法来监测洗脱曲线，分析了在不同条件下纯化的蛋白质 - 洗涤剂复合物的大小、寡聚状态和组成。开发了有效的程序以获得高浓度的纯化TMP，而不过度浓缩洗涤剂。
