Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein
Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein
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DOI：
10.1042/0264-6021:3380305
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发表时间：
1999-03-01
影响因子：
4.1
通讯作者：
Shoulders, CC
中科院分区：
文献类型：
--
作者：
Ritchie, PJ;Decout, A;Shoulders, CC
微粒体甘油三酯转移蛋白（MTP）与蛋白质二硫化物异构酶（PDI）复合，是乳糜微粒和极低密度脂蛋白组装的必需蛋白。MTP-PDI异二聚体的原子结构的测定对于治疗与极低密度脂蛋白的过度产生相关的那些形式的脂蛋白血症具有重要意义，所述脂蛋白易患早发冠心病。为了进行人MTP-PDI复合物的结构研究，有必要产生毫克量的纯蛋白。为此，我们选择了杆状病毒表达系统。用编码FLAG标记的MTP和His标记的PDI的重组病毒共感染昆虫细胞;通过亲和层析纯化所得异源二聚体。从5升昆虫细胞中，获得4-6 mg纯度大于95%的重组蛋白。CD和衰减全反射傅里叶变换红外光谱表明，纯化的蛋白质具有约34%的α-螺旋和33%的β-结构含量。重组蛋白具有与牛MTP-PDI相当的甘油三酯转移活性。描述了针对纯化蛋白的MTP和PDI亚基的多克隆抗体的生产。本研究证明了在昆虫细胞中以高水平表达两种蛋白质的可行性，并描述了用于纯化所得蛋白质复合物的可转移方法。
The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease, To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95 % pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34 % alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.