Choi Ryan, Kelley Angela, Leibly David, Hewitt Stephen Nakazawa, Napuli Alberto, Van Voorhis Wesley
Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Sep 1;67(Pt 9):998-1005. doi: 10.1107/S1744309111017374. Epub 2011 Aug 13.
The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.
可溶性蛋白在大肠杆菌中的重组表达仍然是结构基因组学的一个主要瓶颈。建立用于小规模表达和溶解性测试的可靠方案是结构基因组流程的一个重要组成部分。华盛顿大学的SSGCID蛋白质生产小组（UW-PPG）开发了一种高通量筛选（HTS）方案，用于测量固定化金属亲和色谱（IMAC）中的蛋白质回收率，该方案可预测六组氨酸标签蛋白的成功纯化。该方案基于使用多通道移液器和96孔板手动转移样品，不依赖于机器人平台的使用。该方案已应用于评估在大肠杆菌中表达的4000多种蛋白质的表达和溶解性。UW-PPG还在纯化之前对大规模制备物进行IMAC回收率筛选。对这些结果的分析表明，我们的低成本非自动化方法是满足大型结构基因组项目典型高通量筛选需求的可靠方法。本文详细描述了这些方案以及SSGCID筛选结果的统计分析。结果表明，在小规模和大规模表达后筛选IMAC回收率高的蛋白质，可改善对能够成功纯化并产生晶体结构的蛋白质的选择。
