Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20–100 different constructs followed by large-scale purification of at least 4–6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.
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This project was supported by the Federal Ministry of Economy, Family and Youth through the initiative “Laura Bassi Centres of Expertise”, funding Center of Optimized Structural Studies, project Number 253275; JK was also supported by the Austrian Science Fund (FWF) Project P22276. FWF Doctoral program BioToP-Molecular Technology of Proteins (W1224) is acknowledged to providing training for GM. We thank Dr. Nathalie Landstetter at QIAGEN for generously providing the use of the instrument and valuable suggestions and help with experiments.
Conflict of interest
The authors declare that they have no conflict of interest.
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Mlynek, G., Lehner, A., Neuhold, J. et al. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein–protein complexes. Amino Acids 46, 1565–1582 (2014). https://doi.org/10.1007/s00726-014-1699-x