Recombinant protein expression and solubility screening in Escherichia coli:: a comparative study
Recombinant protein expression and solubility screening in Escherichia coli:: a comparative study
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DOI：
10.1107/s0907444906031337
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发表时间：
2006-10-01
期刊：
影响因子：
--
通讯作者：
Busso, Didier
中科院分区：
文献类型：
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作者：
Berrow, Nick S.;Buessow, K.;Busso, Didier
在大肠杆菌中生产可溶性蛋白质仍然是结构蛋白质组学的主要瓶颈。因此，小规模筛选可溶性表达是鉴定可能适合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) 联盟已经开发了各种表达筛选方法，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协议进行了基准测试。在本研究中，通过大肠杆菌中的表达筛选评估了一组常见的 96 个 His(6-) 标签蛋白的溶解度曲线。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。通过参考蛋白质的子集，证明小规模结果可以提供可能大规模产生的可溶性蛋白质的量的有用指标（即足以进行结构研究）。一般来说，不同群体对于哪些靶标不可溶、哪些靶标最可溶达成了一致。然而，对于大量目标，不同筛选方法报告的结果存在很大差异，这与所探索的程序和参数范围的变化相关。在资源有限的情况下，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。
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. Avariety 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.