The SMALL-SCALE EXPRESSION TESTING (SSET) Team was headed by Brian Fox, PhD, and was responsible for small-scale protein expression trials. Trials were used by staff to evaluate the use of lowered temperature, different medium compositions, and different host strains on protein expression and stability.
One of the main goals of the NIH Protein Structure Initiative was to develop high-throughput technologies to assist protein structure determination. CESG has developed a rapid, small-scale, high-throughput screening method for identifying positively expressed cloned eukaryotic genes (from Arabidopsis thaliana, rice, human, yeast, zebrafish, mouse, and others) suitable for downstream large-scale protein production and subsequent protein purification efforts. The genes are produced with maltose-binding protein (MBP) as an N-terminal fusion to enhance solubility and folding.
CESG small-scale methodologies included 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. High-throughput processing of workgroups of 96 eukaryotic cloned genes are processed and screened in parallel, on the small-scale, for positive expression, solubility, and TEV protease cleavage using the E. coli host strain B834-pRARE2. Large-scale growths are used to produce isotopically enriched proteins for IMAC purification leading to samples for structural analysis by NMR spectroscopy and X-ray crystallography.
The CESG cell-based small-scale methodologies proved 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 mentione properties are made prior to committing valuable resources for downstream processes. Over a two-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.
CESG’s strategy is to minimize costs and maximize the output of soluble proteins suitable for purification uses small-scale screening methodologies to identify protein 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.