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Expression and refolding of recombinant human alpha-tocopherol transfer protein capable of specific alpha-tocopherol binding

机译:具有特异性α-生育酚结合能力的重组人α-生育酚转移蛋白的表达和重折叠

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alpha-Tocopherol transfer protein (alpha-TTP) is a cytosolic protein found predominantly in mammalian liver that is proposed to be responsible for the stereoselective uptake of alpha-tocopherol from the diet. Although recombinant alpha-TTP has been reported previously, little detail has been provided about the yields and competency of the recovered protein at binding tocopherols and other ligands. In this work, we report the successful expression and refolding of a recombinant human alpha-TTP. Ligation-independent cloning generated a construct in pET-30 encoding an alpha-TTP fusion protein (pET-30/ttp) containing a six-histidine tag and an S-tag, each cleavable by a separate protease upon expression in Escherichia coli. Overexpression of the protein led to the formation of inclusion bodies that were solubilized in 8 M urea and purified by metal chelate affinity chromatography. Another construct in pET-28b (pET28b/ttp) provided a soluble protein product after expression that contained a 40-amino-acid N-terminal extension, which can be reduced to 21 amino acids by cleavage with thrombin. The success of different refolding experiments was assessed using a Lipidex gel-based tocopherol binding assay. The best recovery of refolded recombinant alpha-TTP fusion capable of binding a-tocopherol was provided by matrix-assisted refolding in the presence of 0.5 M arginine. Cleavage of the fusion protein with Factor Xa successfully generated the full-length wild-type protein with no additional N-terminal amino acids. The resulting purification scheme provides recombinant alpha-TTP in good yield and purity for investigation of both its structure and its binding affinities for different ligands including natural and synthetic tocols. (C) 2002 Elsevier Science (USA). [References: 34]
机译:α-生育酚转移蛋白(α-TTP)是一种主要在哺乳动物肝脏中发现的胞质蛋白,被认为与饮食中α-生育酚的立体选择性摄取有关。尽管先前已经报道了重组α-TTP,但是几乎没有提供关于回收蛋白在结合生育酚和其他配体上的产量和能力的详细信息。在这项工作中,我们报告了重组人α-TTP的成功表达和重折叠。独立于连接的克隆在pET-30中产生了一个构建体,该构建体编码包含六个组氨酸标签和一个S标签的α-TTP融合蛋白(pET-30 / ttp),当在大肠杆菌中表达时,它们可以被一个单独的蛋白酶切割。蛋白质的过表达导致包涵体的形成,将其溶于8 M尿素中并通过金属螯合亲和层析纯化。 pET-28b中的另一种构建体(pET28b / ttp)在表达后提供了一种可溶性蛋白质产物,该产物包含40个氨基酸的N端延伸,通过凝血酶裂解可将其还原为21个氨基酸。使用基于Lipidex凝胶的生育酚结合测定法评估了不同重新折叠实验的成功性。在0.5 M精氨酸存在下,通过基质辅助的重折叠可以使能够结合α-生育酚的重折叠重组α-TTP融合蛋白得到最佳回收。用因子Xa切割融合蛋白成功地产生了全长野生型蛋白,而没有额外的N端氨基酸。所得的纯化方案以良好的产率和纯度提供了重组α-TTP,用于研究其结构以及对不同配体(包括天然和合成母育酚)的结合亲和力。 (C)2002 Elsevier Science(美国)。 [参考:34]

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