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首页> 外文期刊>Crystal growth & design >Purification, refolding, crystallization and diffraction analysis of the native and selenomethionine-substituted rat Epidymal-Specific lipocalint
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Purification, refolding, crystallization and diffraction analysis of the native and selenomethionine-substituted rat Epidymal-Specific lipocalint

机译：天然和硒代蛋氨酸取代的大鼠附睾特异性脂蛋白的纯化，复性，结晶和衍射分析

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摘要

We describe a straightforward crystallogenesis protocol leading to the preparation of protein crystals suitable for structure determination that involves protein expression and purification, refolding of the overexpressed protein, search of optimal crystallization conditions and diffraction data collection on native and selenomethionine substituted-crystals. The protocol is exemplified with epididymal-specific lipocalin (rLcn6), a newly discovered monomeric protein of 19 kDa that may play an important role in sperm maturation. This protein was cloned from Norway rat (Rattus norvegicus) genome and expressed as insoluble inclusion bodies in Escherichia coli. After refolding of the purified protein, microcrystals were obtained after sparse matrix screening. Optimization of conditions after stepwise incremental initial conditions led to single crystals belonging to space group P2(1)2(1)2(1) and data set at 1.90 angstrom. After attempts with several models, initial phasing was not found by molecular replacement. A similar methodological scheme was used to grow quality crystals of the selenomethionine-substituted rLcn6 protein and collect diffraction data at 2.0 angstrom, allowing phasing and structure resolution. This protocol may be of particular help when overproduction results in denatured proteins within inclusion bodies, a situation that often occurs especially with proteins from eukaryotes, as well as with structural genomic projects.
机译：我们描述了一种简单的结晶发生方案，可导致制备适合于结构确定的蛋白质晶体，涉及蛋白质的表达和纯化，过表达蛋白质的折叠，最佳结晶条件的搜索以及天然和硒代蛋氨酸取代晶体的衍射数据收集。该方案以附睾特异的脂蛋白（rLcn6）为例，这是一种新发现的19 kDa单体蛋白，可能在精子成熟中起重要作用。该蛋白是从挪威大鼠（Rattus norvegicus）基因组克隆而来，并在大肠杆菌中表达为不溶性包涵体。重新折叠纯化的蛋白质后，在稀疏基质筛选后获得微晶。逐步增加初始条件后的条件优化导致单晶属于空间群P2（1）2（1）2（1），数据设置为1.90埃。在尝试了几种模型后，分子置换未发现初始阶段。使用类似的方法学方案来生长硒甲硫氨酸取代的rLcn6蛋白的优质晶体，并收集2.0埃的衍射数据，从而可以定相并进行结构拆分。当过量生产导致包涵体内的蛋白质变性时，该方案可能会特别有用，这种情况尤其会发生在真核生物的蛋白质以及结构基因组计划中。

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