Berrow Nick S, Büssow K, Coutard B, Diprose J, Ekberg M, Folkers G E, Levy N, Lieu V, Owens R J, Peleg Y, Pinaglia C, Quevillon-Cheruel S, Salim L, Scheich C, Vincentelli R, Busso Didier
Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, Oxford, England.
Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1218-26. doi: 10.1107/S0907444906031337. Epub 2006 Sep 19.
Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.
在大肠杆菌中表达可溶性蛋白质仍然是结构蛋白质组学的一个主要瓶颈。因此，小规模筛选可溶性表达是鉴定可能适合进行结构分析的构建体的一种有吸引力的方法。欧洲结构蛋白质组学（SPINE）联盟已经开发了多种表达筛选方法，为了帮助进一步完善这些方法，参与该网络的八个实验室对其方案进行了基准测试。在本研究中，通过在大肠杆菌中进行表达筛选，评估了一组共96种带有His(6)标签的蛋白质的溶解度谱。根据估计的蛋白质产量对每个靶标的可溶性表达水平进行评分。通过参考一部分蛋白质，证明小规模结果可以为大规模生产（即足以用于结构研究）的可溶性蛋白质数量提供有用的指标。一般来说，不同组之间对于哪些靶标不溶以及哪些靶标最溶存在共识。然而，对于大量靶标，不同筛选方法报告的结果存在很大差异，这与实验步骤的差异以及所探索参数的范围有关。鉴于资源有限，如何最有效地探索“表达空间”的问题似乎与晶体学家面临的其他几个多参数问题类似，例如结晶。
