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

BMC Structural Biology

Anne Diehl¹  Konrad Büssow²  Ivica Letunic³  Bernd Simon³  Brigitte Schlegel¹  Martina Leidert¹  Volker Sievert²  Dietmar Leitner¹  Christoph Scheich² 

[1] Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany;Max Planck Institut für Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany;European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany

关键词: high-throughput expression;    protein domains;    homonuclear NMR;    hydrophobic interaction chromatography;    structural genomics;   

Others  :  1181465
DOI  :  10.1186/1472-6807-4-4

 received in 2004-01-06, accepted in 2004-03-08,  发布年份 2004

【 摘 要 】

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.

【 授权许可】

   
2004 Scheich et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

【 图 表 】

Figure 1.

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