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首页> 外文期刊>Infection and immunity >Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli
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Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

机译：大肠杆菌中表达的恶性疟原虫顶膜抗原1的重折叠电子域的纯化，表征和免疫原性。

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The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83Gly to 531Glu) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.
机译：根尖膜抗原1（AMA1）已成为抗疟疾的有希望的疫苗候选者。要进一步评估其对人体的保护功效，就需要生产高度纯化和正确折叠的蛋白质。我们在这里描述了恶性疟原虫AMA1重组胞外域（氨基酸83 Gly 至531 Glu ）的表达，发酵，重折叠和纯化的过程（3D7）在大肠杆菌中产生。包含 E的合成基因。将大肠杆菌密码子偏好性克隆到修饰的pET32质粒中，并用氧化还原修饰的 E产生重组蛋白。大肠杆菌折纸（DE3）。开发了一种纯化方法，其中包括Sarkosyl提取，然后在Ni-三硝基三乙酸柱上进行亲和纯化。在存在还原和氧化的谷胱甘肽的情况下，将重组AMA1重新折叠，并通过两个离子交换色谱步骤进一步纯化。最终产品命名为AMA1 / E，是均匀的，单体的，纯度> 99％，内毒素含量低，宿主细胞污染低。 AMA1 / E的分析表明它具有预测的一级序列，三级结构分析证实了其紧密的二硫键键合性质。针对该蛋白的兔抗体识别天然寄生虫AMA1，并抑制了 P的生长。恶性疟原虫同源3D7克隆的体外试验。已显示AMA1 / E上的还原敏感性表位对于产生抑制性抗AMA1抗体是必需的。 AMA1 / E被构象依赖性生长抑制单克隆抗体4G2dc1识别。本文所述的方法已成功扩大规模，以在GMP条件下生产AMA1 / E蛋白，并且发现该产品在兔中诱导了高度抑制性抗体。

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