A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins
A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins
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DOI：
10.1016/j.molbiopara.2011.04.005
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发表时间：
2011-07-01
影响因子：
1.5
通讯作者：
LaCount, Douglas J.
中科院分区：
文献类型：
--
作者：
Brown, Hakeenah F.;Wang, Ling;LaCount, Douglas J.
使用酵母双杂交测定法研究恶性疟原虫蛋白质-蛋白质相互作用受到恶性疟原虫基因在酵母中表达差和缺乏容易实施的测定法来确认结果的限制。我们在这里报告了两种方法来创建基因片段-随机片段的部分DNA酶I消化和生成密集重叠的片段通过PCR -使大多数部分的恶性疟原虫基因的酵母双杂交试验中表达和筛选。基于PCR的方法在技术上挑战性较小，并且有助于蛋白质相互作用结构域的精细映射。两种方法均揭示了PfMyb 2（PF10_0327）和PFC 0365 w之间的假定相互作用。我们开发了新的质粒来表达小麦胚芽提取物中的蛋白质，并证实了分裂荧光素酶测定和谷胱甘肽-S-转移酶和HA标记的蛋白质的共纯化实验中的相互作用。改进的酵母双杂交筛选方法和验证相互作用的方便系统的组合增强了酵母双杂交测定法用于恶性疟原虫的实用性。(C)2011 Elsevier B. V.保留所有权利。
Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum. (C) 2011 Elsevier B.V. All rights reserved.