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首页> 外文期刊>Protein Expression and Purification >Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+-ATPase
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Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+-ATPase

机译：带有钙调蛋白结合结构域的重组膜蛋白的纯化：大肠杆菌烟酰胺核苷酸转氢酶和人质膜Ca2 + -ATPase C端嵌合体的特性

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摘要

A Ca2+-dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/ CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+-ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 aminio acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4 to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6 x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general. (C) 2004 Elsevier Inc. All rights reserved.
机译：Ca2 +依赖性钙调蛋白结合肽（CBP）是用于重组蛋白（尤其是膜蛋白）亲和纯化的有吸引力的标签，因为洗脱是通过去除/螯合Ca2 +来简单完成的。为了开发用于大肠杆菌烟酰胺核苷酸转氢酶的一步调节钙调蛋白/ CBP依赖的纯化程序，将人质膜Ca2 + -ATPase（hPMCA）的49个氨基酸的大CBP或更大的149个氨基酸的C末端片段融合了C-末端位于转氢酶的β亚基上。使用49个氨基酸片段的融合导致转氢酶表达的显着损失，而与149个氨基酸片段的融合给出令人满意的表达。该嵌合蛋白通过在钙调蛋白-琼脂糖上的亲和色谱纯化，并用EDTA温和洗脱。纯度和活​​性与用His标记的转氢酶获得的纯度和活性相当，并显示出增加的稳定性。与野生型转氢酶相比，带有CBP标签的转氢酶相对于β亚基的α亚基含量高4至10倍。为了确定后者是否是由于CBP标签引起的，使用N-末端6 x His标签和CBP标签（通过使用任一标签进行纯化）的双标签转氢酶与CBP标签相比，纯度没有显着提高。单标签蛋白。讨论了亚基组成改变的原因。结果表明，取决于构建体，CBP标签通常可以是适合于膜蛋白的亲和纯化标签。 （C）2004 Elsevier Inc.保留所有权利。

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