Preparation of cytochromes b5 with an extended COOH-terminal hydrophilic segment: Interaction of modified tail-anchored proteins with liposomes in different cholesterol content

Y. Sakamoto, F. Takeuchi, M. Miura, Sam-Yong Park, M. Tsubaki
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Abstract

A group of membrane proteins endowed with a single COOH-terminal hydrophobic domain capable of insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b5 (Hcytb5) having various COOH-terminal extensions with bovine opsin sequence, we constructed expression vectors containing membrane-bound form of Hcytb5 (or Hcytb5op(a), Hcytb5op(b), Hcytb5op(c), and Hcytb5op(d), where NH2-terminal bovine opsin sequences with various truncated forms were attached at the COOH-terminus) and established a method for their expression and purification in the holo-form. We analyzed the integration of the TA domain of holo-form of Hcytb5 into protein-free liposomes. The integration of holo- Hcytb5 occurred efficiently into the membranes with a low cholesterol content even in the presence of a small amount of Triton X−100 and, once incorporated, the proteoliposomes were relatively stable.
带扩展cooh末端亲水段的细胞色素b5的制备:不同胆固醇含量的修饰尾锚定蛋白与脂质体的相互作用
一组具有单一cooh末端疏水结构域的膜蛋白被称为尾部锚定(TA)蛋白。为了明确具有不同cooh末端延伸的人细胞色素b5 (Hcytb5) ta结构域与牛视蛋白序列的插入机制,我们构建了含有膜结合形式的Hcytb5(或Hcytb5op(a)、Hcytb5op(b)、Hcytb5op(c)和Hcytb5op(d)的表达载体,其中不同截断形式的nh2末端牛视蛋白序列连接在cooh末端),并建立了其全形式表达和纯化方法。我们分析了Hcytb5全息形式的TA结构域与无蛋白脂质体的整合。即使在少量Triton X - 100存在的情况下,holo- Hcytb5也能有效地整合到低胆固醇含量的膜中,并且一旦整合,蛋白脂质体相对稳定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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