Abstract
BackgroundAn arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.ResultsA robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25°C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.ConclusionSoluble fusion proteins can be produced in E. coli in sufficient qualities and μg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.
Highlights
An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis
The resulting in-vitro data could help to bridge the knowledge from different largescale technologies for functional genomics and proteomics applications [21,22]
Technical set-up of the fully automated system The liquid handling steps required for open reading frames (ORF) cloning, protein expression and protein purification were implemented on the MULTI-probe II robot which was controlled with the application system software, if possible
Summary
An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The baculovirus as well as yeast expression systems have been exploited to produce proteins on a large scale for subsequent production of microarrays [18]. Both expression systems introduce host-specific post-translational modifications. The bacterial expression system Escherichia coli [19] produces proteins devoid of those post-translational modifications typically present in endogenously expressed mammalian proteins. This circumstance can be advantageous for certain applications, e.g. to screen for novel substrates of human kinases. The resulting in-vitro data could help to bridge the knowledge from different largescale technologies for functional genomics and proteomics applications [21,22]
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