Expression of biologically active rat apolipoprotein AIV in Escherichia coli
Expression of biologically active rat apolipoprotein AIV in Escherichia coli
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DOI：
10.1016/s0031-9384(02)00959-9
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发表时间：
2003-01-01
影响因子：
2.9
通讯作者：
Tso, P
中科院分区：
文献类型：
--
作者：
Liu, M;Maiorano, N;Tso, P
大鼠载脂蛋白AIV(Apo AIV)是一种43 kDa的肠道载脂蛋白，在脂代谢和抑制食物摄入中起重要作用。在本研究中，大鼠载脂蛋白AIV全长在大肠杆菌中表达，并以生物活性形式纯化。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和质谱分析表明，重组蛋白的相对分子质量约为43 kDa，与天然大鼠apo AIV免疫印迹分析和N端氨基酸序列测定证实重组蛋白为天然大鼠apo AIV。重组蛋白具有与脂蛋白结合的功能。生物学活性的评估是，与天然大鼠apo AIV相比，重组蛋白抑制了禁食大鼠的食物摄入量，在抑制摄食的剂量下，天然或重组apo AIV都不会引起条件性味觉厌恶(CTA)。这些结果表明，重组apo AIV在结构和功能上与大鼠天然apo AIV没有区别，这使得这种高表达和纯化方案成为未来结构和功能研究的有力工具。(C)2003 Elsevier Science Inc.保留所有权利。
Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies. (C) 2003 Elsevier Science Inc. All rights reserved.