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 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.


CONCLUSIONS
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.
Scheich
Christoph
C
Proteinstrukturfabrik, Heubnerweg 6, 14059 Berlin, Germany. scheich@molgen.mpg.de
Leitner
Dietmar
D
Sievert
Volker
V
Leidert
Martina
M
Schlegel
Brigitte
B
Simon
Bernd
B
Letunic
Ivica
I
Büssow
Konrad
K
Diehl
Anne
A
D002845:Chromatography; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D030562:Databases, Protein; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D013997:Time Factors
D011506:Proteins