SMALL-SCALE HIGH-THROUGHPUT SCREEN OF EUKARYOTIC PROTEIN PRODUCTION

CESG's strategy to minimize costs and maximize the output of soluble proteins suitable for purification uses small-scale screening methodologies to identify proteins targets that are statistically likely to proceed through all downstream processing stages (large-scale protein production and generic IMAC purification). Passage through the entire cell-based protein production process, beginning with gene cloning, requires ~2-3 months to deliver ~10 mg of purified, isotopically enriched protein for either NMR spectroscopy screens or X-ray crystallization trials. Small-scale screening identifies several important features of cloned eukaryotic proteins that together indicate whether the proteins will be "suitable" for structural studies. CESG small-scale methodologies include expression vector engineering, optimization and improvement of auto-induction medium using factorial evolution techniques, and automated protein production analysis. These approaches have been used to identify the best eukaryotic proteins (Arabidopsis, rice, human, yeast, zebrafish, mouse, and algae) for large-scale cell growths (2 liter), and also yield sufficient sample for functional studies and preliminary structural analysis. The CESG cell-based small-scale methodologies have proven to be rapid, simple, efficient, and cost-effective. The approaches developed provide information on the level of expression, the potential for in vivo degradation; the solubility of the MBP fusion; the ability of TEV protease to cleave the fusion, and the yield of cleaved and purified target protein.The small-scale screening method is flexible and assessments of the above mentioned properties are made prior to committing valuable resources for downstream processes. Over a 2 year period, this approach has successfully predicted proteins that are suitable for large-scale production and purification with ~80% accuracy. In summary, the CESG small-scale purification screening approach saves considerable resources and labor, and reduces expenditure of resources on proteins targets that will ultimately fail in protein purification.