Isolation and refolding of H-ras from inclusion bodies of Escherichia coli: refold procedure and comparison of refolded and soluble H-ras
| Autor: | D E Van Dyk, T E Van Aken, S. L. Campbell-Burk, R J DeLoskey |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
Glycerol
Protein Folding Magnetic Resonance Spectroscopy Biophysics Glycine medicine.disease_cause Biochemistry High-performance liquid chromatography Guanosine Diphosphate Inclusion bodies Proto-Oncogene Proteins p21(ras) chemistry.chemical_compound medicine Escherichia coli Serine Molecular Biology Chromatography High Pressure Liquid Chromatography Ion exchange Chemistry Temperature Nuclear magnetic resonance spectroscopy Chromatography Ion Exchange Solubility Yield (chemistry) Protein folding Guanosine Triphosphate |
| Zdroj: | Archives of biochemistry and biophysics. 311(1) |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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