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The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein–protein complexes

Amino Acids volume 46, pages 1565–1582 (2014)Cite this article

Abstract

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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Acknowledgments

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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Author notes

  1. Nikos Pinotsis

    Present address: Birkbeck College, University of London, Malet Street, London, WC1E 7HX, UK

  2. Anita Lehner, Jana Neuhold & Peggy Stolt-Bergner

    Present address: Protein Technologies Facility, Campus Science Support Facilities GmbH, Dr. Bohrgasse 3, 1030, Vienna, Austria

Authors and Affiliations

  1. Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria

    Georg Mlynek, Anita Lehner, Jana Neuhold, Sarah Leeb, Julius Kostan, Alexej Charnagalov, Kristina Djinović-Carugo & Nikos Pinotsis

  2. Research Institute of Molecular Pathology, Dr. Bohr-gasse 7, 1030, Vienna, Austria

    Peggy Stolt-Bergner

  3. Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000, Ljubljana, Slovenia

    Kristina Djinović-Carugo

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  1. Georg Mlynek
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Correspondence to Kristina Djinović-Carugo or Nikos Pinotsis.

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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

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