Methodology article

Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

Christoph Scheich^1,2*†, Dietmar Leitner^3,1†, Volker Sievert^1,2, Martina Leidert³, Brigitte Schlegel³, Bernd Simon^4,1, Ivica Letunic⁴, Konrad Büssow^1,2 and Anne Diehl^3,1

• * Corresponding author: Christoph Scheich scheich@molgen.mpg.de

• † Equal contributors

Author Affiliations

¹ Proteinstrukturfabrik, Heubnerweg 6, 14059 Berlin, Germany

² Max Planck Institut für Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany

³ Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany

⁴ European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany

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BMC Structural Biology 2004, 4:4 doi:10.1186/1472-6807-4-4

Published: 8 March 2004

Abstract

Background

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.

Results

88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D ¹H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC) was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains) were quickly identified as being well folded and suitable for structural analysis.

Conclusion

The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D ¹H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.