艰难梭菌PPEP-1通过微量接种和锌单波长反常散射法的制备、结晶及结构测定
Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD.
作者信息
Pichlo Christian, Montada Angelika A, Schacherl Magdalena, Baumann Ulrich
机构信息
Institute of Biochemistry, University of Cologne.
Institute of Biochemistry, University of Cologne;
出版信息
J Vis Exp. 2016 Dec 30(118):55022. doi: 10.3791/55022.
Abstract
New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.
摘要
需要新的疗法来治疗艰难梭菌感染，这种感染对人类健康构成重大威胁。艰难梭菌金属蛋白酶PPEP-1是未来开发抑制剂以降低病原体毒力的靶点。为了进行生物物理和结构表征以及抑制剂筛选，将需要大量的纯活性蛋白。我们已经开发了一种方案，通过使用大肠杆菌作为表达宿主来高效生产和纯化PPEP-1，从而获得足够数量和纯度的蛋白质用于结晶和结构测定。此外，通过微种晶法，可以将高度共生生长的PPEP-1晶体生长成适合X射线衍射分析的有序晶体。这些方法也可用于生产其他重组蛋白，并研究产生共生晶体的其他蛋白的结构。
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