Abstract
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.
Highlights
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
Small-scale screening for soluble expression in Escherichia coli is a key feature of the experimental pipelines that have been implemented for structural proteomics projects in a number of European laboratories (Alzari et al, 2006)
The central question is ‘how should we configure small-scale expression screening in order to establish a route to the production of milligram amounts of soluble protein on scale-up?’ To this end, we have compared the methods routinely used in eight different laboratories in the SPINE consortium
Summary
Small-scale screening for soluble expression in Escherichia coli is a key feature of the experimental pipelines that have been implemented for structural proteomics projects in a number of European laboratories (Alzari et al, 2006). The justification for such screening is that it provides information to guide subsequent decisions on whether to invest in largescale purification of a given construct. Screening methods are only of value if they satisfy two fundamental criteria They must be reproducible and secondly, they must give reliable qualitative and ideally quantitative predictions of the outcome of the larger scale protein production needed to produce sufficient protein for structural studies. The central question is ‘how should we configure small-scale expression screening in order to establish a route to the production of milligram amounts of soluble protein on scale-up?’ To this end, we have compared the methods routinely used in eight different laboratories in the SPINE consortium
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