Gosavi Rajendrakumar A, Mueser Timothy C, Schall Constance A
Department of Chemical Engineering, The University of Toledo, Toledo, OH 43606, USA.
Acta Crystallogr D Biol Crystallogr. 2008 May;64(Pt 5):506-14. doi: 10.1107/S0907444908004265. Epub 2008 Apr 19.
Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.
将蛋白质储备溶液的溶解度提高到高于标准色谱缓冲液（50 mM Tris-HCl，pH 7.5，100 mM NaCl）中的溶解度，使得接受两种晶体筛选（即索引筛选和沉淀剂/沉淀剂添加剂（P/PA）筛选）的十种球状蛋白质的结晶条件数量增加。通过筛选和选择缓冲液成分来配制最佳缓冲液，实现了蛋白质储备溶液溶解度的提高。通过防止聚乙二醇8000导致蛋白质沉淀来估计溶解度的相对提高。在最佳缓冲液中溶解度提高有限的蛋白质在添加甘油后溶解度增强。然后通过优化溶液的浓度确定最大溶解度，直至形成沉淀。上清液浓度随后提供了蛋白质溶解度上限的估计值。这个“溶解度”估计值用于确定筛选实验中所用蛋白质的初始浓度，并且是成功结晶的重要一步。基于溶解度估计进行缓冲液优化和确定晶体筛选的初始蛋白质浓度，为改善晶体筛选结果提供了一种方法。
