A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase
A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase
复制标题
DOI：
10.1110/ps.8.7.1475
复制
发表时间：
1999-07-01
期刊：
影响因子：
8
通讯作者：
Gouaux, E
中科院分区：
文献类型：
--
作者：
Armstrong, N;De Lencastre, A;Gouaux, E
从大肠杆菌包涵体中生产折叠的具有生物活性的蛋白质只有在体外自然化的方案存在的情况下才能实现。由于需要一种快速和有统计学意义的方法来确定和评估蛋白质折叠条件，我们设计了一种新的分数因子蛋白质折叠筛选。该筛选包括先前实验所示的增强蛋白质折叠的12个因子，并将这12个因子纳入16种不同的折叠条件中。通过检查全阶乘的1/256（th）部分，确定了谷氨酸和红藻氨酸受体的配体结合结构域以及溶菌酶和碳酸酐酶B的多重折叠条件。通过计算因子主效应估计每个因子对生物活性物质形成的影响。因素和相应的水平，如pH值（8.5）和L-精氨酸（0.5 M）始终对蛋白质折叠有积极的影响，而洗涤剂（0.3 mM月桂基麦芽糖苷）和非极性添加剂（0.4 M蔗糖）对这四种蛋白质的折叠是有害的。16种条件中的一种产生了四种蛋白质中三种的最折叠材料。我们的研究结果表明，这种蛋白质折叠筛选通常有助于确定其他蛋白质是否会在体外折叠，如果是这样，哪些因素是重要的。此外，分数析因折叠筛选非常适合于评估以前未测试的蛋白质折叠因子。
Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 1/256(th) fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.