Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance
Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance
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DOI：
10.1002/jmr.2292
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发表时间：
2013-10-01
影响因子：
2.7
通讯作者：
Bosch, Juergen
中科院分区：
文献类型：
--
作者：
Boucher, Lauren E.;Bosch, Juergen
我们已经开发了一种表达系统，能够生产大量的低成本，特定的肽，无论是His（12）-标记，生物素化，或未标记。该肽系统的灵活性适合于通过表面等离子体共振（SPR）、共结晶和酶联免疫吸附测定进行相互作用研究。将含有除15个氨基酸的AviTag之外的感兴趣的肽序列的基因块克隆到表达N-末端麦芽糖结合蛋白的载体中。表达并纯化构建体，并通过分析性尺寸排阻色谱法估计重组蛋白的分子量。通过抗生物素蛋白质印迹证实AviTag的成功原位生物素化，并用于偶联至表面等离子体共振芯片。我们能够验证，作为概念研究的证据，恶性疟原虫醛缩酶（PfAldolase）与血小板反应蛋白相关的无名蛋白（TRAP）家族的三种不同的细胞质粘附素尾肽的特异性蛋白质-蛋白质相互作用，并确定它们的亲和力。这种肽生产方法能够以两天、成本有效的方式高产量地生产肽。该工具将使我们能够筛选针对疟原虫物种中的滑体的肝脏阶段和血液阶段复合物的蛋白质-蛋白质相互作用抑制剂。这一工具的适应将使研究人员能够继续自己的蛋白质-蛋白质相互作用的研究。版权所有（c）2013约翰威利父子有限公司
We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His(12)-tagged, biotinylated, or unlabeled. The flexibility of this peptide system is suitable for interaction studies via surface plasmon resonance (SPR), co-crystallization, and enzyme-linked immunosorbent assay. Gene blocks containing peptide sequences of interest in addition to a 15 amino acid AviTag, were cloned into a vector expressing an N-terminal maltose binding protein. The constructs were expressed and purified, and the molecular weights of the recombinant proteins were estimated by analytical size exclusion chromatography. Successful in situ biotinylation of the AviTag was confirmed by anti-biotin western blot and was used for coupling to the surface plasmon resonance chip. We were able to validate, as a proof of concept study, the specific protein-protein interaction of Plasmodium falciparum aldolase (PfAldolase) with three different cytoplasmic adhesin tail peptides from the family of thrombospondin-related anonymous proteins (TRAPs), and to determine their affinities. This method of peptide production enables high yield production of peptides in a two-day, cost effective manner. This tool will allow us to screen for protein-protein interaction inhibitors directed toward the liver stage and blood stage complexes of the glideosome in Plasmodium species. Adaptation of this tool will allow researchers to pursue their own studies of protein-protein interactions. Copyright (c) 2013 John Wiley & Sons, Ltd.