Isolation and Refolding of H-ras from Inclusion Bodies of Escherichia coli: Refold Procedure and Comparison of Refolded and Soluble H-ras

A refolding and purification method for economically producing large quantities of H-ras isolated from Escherichia coli inclusion bodies is described. Experiments were performed to optimize the yield of refolded H-ras for structural analysis by NMR spectroscopy. Protein concentration, temperature, and the presence of 10% glycerol during refolding were varied. The yield of H-ras was highest when the protein was refolded at concentrations less than or equal to 0.1 mg/ml and was independent of the presence of 10% glycerol. The yield was slightly higher at 4°C than at 25°C. The refolded H-ras was purified by anion exchange chromatography to yield protein with a purity of >90%, as determined by C4 reverse-phase HPLC, and a GDP-binding stoichiometry greater than 0.9. NMR structural analysis of refolded and soluble H-ras was conducted using [15N] glycine- and [15N]serine-enriched protein. The NMR data indicate that the refolded ras protein is structurally similar to ras isolated from the soluble fraction.