Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands
Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands
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DOI：
10.1177/1087057109335749
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发表时间：
2009-07-01
影响因子：
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通讯作者：
Van Voorhis, Wesley C.
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文献类型：
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作者：
Crowther, Gregory J.;Napuli, Alberto J.;Van Voorhis, Wesley C.
在过去的十年中，热熔体/热移测定法已经成为鉴定配体和其他稳定特定蛋白质的因素的常用工具。蛋白质的熔化温度（T(m)）的增加表明稳定性的增加。在优化后续化合物文库筛选的检测方法时，重要的是尽量减少T(m)测量的可变性，从而最大限度地提高检测潜在配体的能力。作者对疟原虫重组蛋白的T(m)变异性进行了研究。疟原虫蛋白的配体作为疟疾药物的潜在起点尤其有趣，而且迫切需要新的药物。单一标准缓冲液（100 mM HEPES [pH 7.5], 150 mM NaCl）可对61种疟原虫蛋白中的58种进行T(m)估计。然而，对于几种蛋白质，T(m)不能以适合高通量筛选的一致性测量，除非使用替代蛋白质特异性缓冲液。作者得出结论，将T(m)测量的可变性最小化的缓冲液优化提高了涉及蛋白质的热熔筛的成功率，而标准缓冲液是次优的。（Journal of biomolmolecular Screening 2009:700-707）
In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (T(m)). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of T(m) measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of T(m) variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of T(m) for 58 of 61 Plasmodium proteins tested. However, with several proteins, T(m) could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in T(m) measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. (Journal of Biomolecular Screening 2009:700-707)