Abstract
BackgroundThe availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells.Results and ConclusionProtein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins.
Highlights
The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis
This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells
Only partial sequence information existed for these clones – the EST sequences of the dbEST database [34] – and only a small proportion contained complete open reading frames. 490 proteins were selected which met the selection criteria, had no match to Protein Data Bank (PDB) detected by PSI-BLAST and for which full-ORF IMAGE cDNA clones were available
Summary
The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells. The Protein Structure Factory The Protein Structure Factory (PSF) is a joint endeavour of universities, research institutes and companies from the Berlin area [1,2] It takes part in the international structural genomics initiative [3,4] and aims at the determination of human protein structures by X-ray diffraction methods and NMR spectroscopy using standardised highthroughput procedures. Alternative expression systems include cell cultures of various eukaryotic organisms and (page number not for citation purposes)
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