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 degrees C than at 25 degrees 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.
DeLoskey
R J
RJ
Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0228.
Van Dyk
D E
DE
Van Aken
T E
TE
Campbell-Burk
S
S
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000737:chemistry*; D005990:Glycerol / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D012995:Solubility; D013696:Temperature
D006153:Guanosine Diphosphate; D012694:Serine; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras); D005990:Glycerol; D005998:Glycine