Dobrovetsky Elena, Lu Ming Liang, Andorn-Broza Ronit, Khutoreskaya Galina, Bray James E, Savchenko Alexei, Arrowsmith Cheryl H, Edwards Aled M, Koth Christopher M
Ontario Center for Structural Proteomics, University of Toronto, 112 College St., Toronto, Ontario, Canada.
J Struct Funct Genomics. 2005;6(1):33-50. doi: 10.1007/s10969-005-1363-5.
Membrane proteins constitute ~30% of prokaryotic and eukaryotic genomes but comprise a small fraction of the entries in protein structural databases. A number of features of membrane proteins render them challenging targets for the structural biologist, among which the most important is the difficulty in obtaining sufficient quantities of purified protein. We are exploring procedures to express and purify large numbers of prokaryotic membrane proteins. A set of 280 membrane proteins from Escherichia coli and Thermotoga maritima, a thermophile, was cloned and tested for expression in Escherichia coli. Under a set of standard conditions, expression could be detected in the membrane fraction for approximately 30% of the cloned targets. About 22 of the highest expressing membrane proteins were purified, typically in just two chromatographic steps. There was a clear correlation between the number of predicted transmembrane domains in a given target and its propensity to express and purify. Accordingly, the vast majority of successfully expressed and purified proteins had six or fewer transmembrane domains. We did not observe any clear advantage to the use of thermophilic targets. Two of the purified membrane proteins formed crystals. By comparison with protein production efforts for soluble proteins, where approximately 70% of cloned targets express and approximately 25% can be readily purified for structural studies [Christendat et al. (2000) Nat. Struct. Biol., 7, 903], our results demonstrate that a similar approach will succeed for membrane proteins, albeit with an expected higher attrition rate.
膜蛋白约占原核生物和真核生物基因组的30%，但在蛋白质结构数据库中的条目却只占一小部分。膜蛋白的一些特性使其成为结构生物学家颇具挑战性的研究对象，其中最重要的是难以获得足够量的纯化蛋白。我们正在探索表达和纯化大量原核膜蛋白的方法。从大肠杆菌和嗜热栖热菌（一种嗜热菌）中选取了一组280种膜蛋白进行克隆，并检测其在大肠杆菌中的表达情况。在一组标准条件下，约30%的克隆靶点在膜组分中能检测到表达。约22种表达量最高的膜蛋白被纯化出来，通常只需两步色谱法。给定靶点中预测的跨膜结构域数量与其表达和纯化的倾向之间存在明显的相关性。因此，绝大多数成功表达和纯化的蛋白具有六个或更少的跨膜结构域。我们没有观察到使用嗜热靶点有任何明显优势。其中两种纯化的膜蛋白形成了晶体。与可溶性蛋白的蛋白质生产工作相比，约70%的克隆靶点能够表达，约25%的靶点能够很容易地纯化用于结构研究[克里斯滕达特等人（2000年）《自然结构生物学》，7，903]，我们的结果表明，类似的方法对膜蛋白也会成功，尽管预计损耗率会更高。
