Chew Ching Hoong, Lim Yvonne Ai Lian, Chua Kek Heng
School of Biomedicine, Faculty of Health Sciences, Universiti Sultan Zainal Abidin, Kuala Nerus, Terengganu, Malaysia.
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
PeerJ. 2017 Sep 13;5:e3794. doi: 10.7717/peerj.3794. eCollection 2017.
is an obligate intracellular parasite. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., for sporozoite to invade and replicate within hepatocytes in the liver stage and merozoite to penetrate and replicate within erythrocytes in the blood stage. AMA1 has long served as a potent antimalarial drug target and is a pivotal vaccine candidate. A good understanding of the structure and molecular function of this protein, particularly its involvement in host-cell adhesion and invasion, is of great interest and hence it offers an attractive target for the development of novel therapeutics. The present study aims to heterologous express recombinant AMA1 ectodomain of (rPvAMA1) for the selection of binding peptides.
The rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXact system and codon optimized BL21-Codon Plus (DE3)-RIL strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.
The rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.
Phage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.
疟原虫是一种专性细胞内寄生虫。顶膜抗原1（AMA1）是最突出且特征明确的疟疾表面抗原，对于寄生虫-宿主细胞入侵至关重要，即在肝脏阶段子孢子侵入并在肝细胞内复制，以及在血液阶段裂殖子穿透并在红细胞内复制过程中发挥作用。AMA1长期以来一直是一种有效的抗疟药物靶点，也是关键的疫苗候选物。深入了解该蛋白的结构和分子功能，尤其是其在宿主细胞黏附和入侵中的作用，具有重要意义，因此它为新型治疗药物的开发提供了一个有吸引力的靶点。本研究旨在异源表达重组恶性疟原虫AMA1胞外结构域（rPvAMA1）以筛选结合肽。
使用无标签的Profinity eXact系统和密码子优化的BL21-Codon Plus（DE3）-RIL菌株异源表达rPvAMA1蛋白，并通过透析进一步重折叠以实现复性。使用Ph.D.-12随机噬菌体展示文库淘选针对重折叠rPvAMA1的结合肽。
rPvAMA1成功表达并复性，鉴定出三种对rPvAMA1具有亲和力的噬菌体展示十二肽，分别命名为PdV1（DLTFTVNPLSKA）、PdV2（WHWSWWNPNQLT）和PdV3（TSVSYINNRHNL）。在直接噬菌体检测中，即噬菌体ELISA结合检测和蛋白质印迹结合检测中，它们均对rPvAMA1呈现阳性结合信号。
噬菌体展示技术基于一个简单原理实现蛋白质-蛋白质相互作用的映射，即使用展示肽的噬菌体颗粒文库，选择并鉴定与靶蛋白结合的噬菌体克隆。使用CABS-dock网络服务器预测每个选定肽与恶性疟原虫AMA1（蛋白质数据库，PDB ID：1W8K）的结合位点。在这种情况下，结合肽为开发针对PvAMA1的拟肽抗疟拮抗剂提供了有价值的起点。
