Recombinant protein expression and solubility screening in Escherichia coli: a comparative study

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 62; no. 10; pp. 1218 - 1226
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Blackwell Publishing Ltd 01.10.2006
• Subjects: Algorithms; Culture Media; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic Vectors; high throughput; Medicin och hälsovetenskap; protein expression; protein production; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Reproducibility of Results; Solubility; Temperature
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444906031337

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 His6‐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.