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

Scheich, C., Leitner, D., Sievert, V., Leidert, M., Schlegel, B., Simon, B., et al. (2004). Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis. BMC Structural Biology, 4, 4-4. doi:doi:10.1186/1472-6807-4-4.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-88AF-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-88B0-1
Genre: Journal Article

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 Creators:
Scheich, Christoph1, Author
Leitner, Dietmar, Author
Sievert, Volker2, Author              
Leidert, Martina, Author
Schlegel, Brigitte, Author
Simon, Bernd, Author
Letunic, Ivica, Author
Büssow, Konrad2, Author              
Diehl, Anne, Author
Affiliations:
1Max Planck Society, ou_persistent13              
2Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              

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Free keywords: structural genomics; hydrophobic interaction chromatography; homonuclear NMR; protein domains; high-throughput expression
 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.

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Language(s): eng - English
 Dates: 2004-03-08
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: eDoc: 230796
DOI: doi:10.1186/1472-6807-4-4
 Degree: -

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Title: BMC Structural Biology
Source Genre: Journal
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Pages: - Volume / Issue: 4 Sequence Number: - Start / End Page: 4 - 4 Identifier: -