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首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >Contamination from an affinity column: An encounter with a new villain in the world of membrane-protein crystallization
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Contamination from an affinity column: An encounter with a new villain in the world of membrane-protein crystallization

机译：污染一个亲和力列:一个遇到一个新的恶棍的世界里膜蛋白结晶

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Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 ? resolution of Strep-Tactin in complex with desthiobiotin.
机译：试图结晶AtNTT1,叶绿体ATP / ADP运输机从拟南芥,Strep-Tactin透露一个意想不到的污染物,期间使用的链霉亲和素的一个变体纯化的蛋白质。出现在非常少量,晶体的Strep-Tactin可再生产地成长的AtNTT1解决方案。大肠杆菌和纯化detergent-solubilized膜分数使用基于一个Strep-Tactin亲和色谱法工程链霉亲和素。Strep-Tactin蛋白质纯化方案列从未前面描述的和似乎是特定于膜蛋白随着洗涤剂。筛选了从Strep-Tactin观察Strep-Tactin列使用几个经常使用洗涤剂,说明其可能的作用的污染。在膜蛋白并建议谨慎净化使用Strep-Tactin亲和力列,在洗涤剂是必不可少的组件。蛋白质污染物导致的小晶体结构在1.9吗?与desthiobiotin复杂。

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