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首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >Effects of impurities on membrane-protein crystallization in different systems
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Effects of impurities on membrane-protein crystallization in different systems

机译：杂质对膜蛋白的影响结晶在不同的系统

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摘要

When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein.
机译：当开始一个蛋白质晶体项目时,科学家们正面临着几个未知数。其中这些问题:(i)起始物料的纯度足够吗?(2)哪种类型的结晶实验最有前途的行为吗?净化活性膜蛋白样品结晶试验和高成本与生产这些样品需要一个非常实用的方法。实用的指导方针需要增加膜蛋白结晶的效率。为了解决这些难题,影响常见的各种杂质膜蛋白结晶机制有被调查发现油脂的立方相(LCP)为基础的结晶方法比结晶更健壮在洗涤剂环境中使用蒸汽扩散或microbatch方法的能力容忍污染形式的蛋白质脂质或其他通用膜组件。LCP-based结晶方法产生的晶体光合反应中心(RC)Rhodobacter sphaeroides从样本大量残留杂质。与蛋白质晶体得到污染水平的50%,增加脂质纯样品材料和膜碎片钢筋混凝土的数量或几乎没有影响在LCP-based获得的晶体质量结晶屏幕。这对杂质可能避免需要宽容样品的超高纯度当事业初始结晶筛选试验确定初步的结晶条件可以优化对于一个给定的目标蛋白质。

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