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首页> 外文期刊>Protein Expression and Purification >Production, characterization and crystallization of the Plasmodium falciparum aquaporin
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Production, characterization and crystallization of the Plasmodium falciparum aquaporin

机译：恶性疟原虫水通道蛋白的生产，表征和结晶

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The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A + T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64 mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18 mg/L) and initial attempts at crystallization of the protein yielded several different forms. (C) 2008 Elsevier Inc. All rights reserved.
机译：疟疾的病原体恶性疟原虫具有单一的水甘油糖蛋白（PfAQP），它代表了治疗该疾病的潜在药物靶标。 PfAQP定位在寄生虫膜上，以运输水，甘油，氨和可能的糖酵解中间体。为了设计抑制剂，我们着手确定PfAQP的3D结构，其中要克服的第一个瓶颈是获得足够高产量的重组蛋白。野生型PfAQP基因在表达宿主大肠杆菌和巴斯德毕赤酵母中表达至低水平或无法检测到，这被认为是由于不同的基因组A + T含量和不同的密码子使用所致。因此，产生了两个密码子优化的PfAQP基因。大肠杆菌的Opt-PfAQP仍可能导致折叠问题，但仍未导致高产量。但是，针对巴斯德毕赤酵母进行优化的PfAQP已在巴斯德毕赤酵母中成功表达以用于生产，并在酿酒酵母中成功表达以进行功能研究。在酿酒酵母中，PfAQP介导的甘油运输，但出乎意料的是，水的运输无法得到证实。在巴斯德毕赤酵母中进行高水平的膜定位表达（估计为每升细胞培养物中64 mg PfAQP）后，将PfAQP纯化至均质（18 mg / L），并且最初的蛋白质结晶尝试产生了几种不同形式。 （C）2008 Elsevier Inc.保留所有权利。

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