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

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...

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Autores principales: Scheich, Christoph, Leitner, Dietmar, Sievert, Volker, Leidert, Martina, Schlegel, Brigitte, Simon, Bernd, Letunic, Ivica, Büssow, Konrad, Diehl, Anne
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516802/
https://www.ncbi.nlm.nih.gov/pubmed/15113422
http://dx.doi.org/10.1186/1472-6807-4-4
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author Scheich, Christoph
Leitner, Dietmar
Sievert, Volker
Leidert, Martina
Schlegel, Brigitte
Simon, Bernd
Letunic, Ivica
Büssow, Konrad
Diehl, Anne
author_facet Scheich, Christoph
Leitner, Dietmar
Sievert, Volker
Leidert, Martina
Schlegel, Brigitte
Simon, Bernd
Letunic, Ivica
Büssow, Konrad
Diehl, Anne
author_sort Scheich, Christoph
collection PubMed
description 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 (1)H-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 (1)H-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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spelling pubmed-5168022004-09-14 Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis Scheich, Christoph Leitner, Dietmar Sievert, Volker Leidert, Martina Schlegel, Brigitte Simon, Bernd Letunic, Ivica Büssow, Konrad Diehl, Anne BMC Struct Biol Methodology Article 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 (1)H-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 (1)H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics. BioMed Central 2004-03-08 /pmc/articles/PMC516802/ /pubmed/15113422 http://dx.doi.org/10.1186/1472-6807-4-4 Text en Copyright © 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.
spellingShingle Methodology Article
Scheich, Christoph
Leitner, Dietmar
Sievert, Volker
Leidert, Martina
Schlegel, Brigitte
Simon, Bernd
Letunic, Ivica
Büssow, Konrad
Diehl, Anne
Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title_full Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title_fullStr Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title_full_unstemmed Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title_short Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis
title_sort fast identification of folded human protein domains expressed in e. coli suitable for structural analysis
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516802/
https://www.ncbi.nlm.nih.gov/pubmed/15113422
http://dx.doi.org/10.1186/1472-6807-4-4
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