Purification and structural characterization of recombinant rat γ-interferon from Escherichia coli
| Autor: | Elizabeth Dharm, Chun-Yen Lai, Yutaka Fujii |
|---|---|
| Rok vydání: | 1989 |
| Předmět: |
Ammonium sulfate
Biophysics medicine.disease_cause Biochemistry Propanol Interferon-gamma chemistry.chemical_compound Adsorption Escherichia coli medicine Animals Chemical Precipitation Amino Acid Sequence Molecular Biology Chromatography Elution Extraction (chemistry) Rats Inbred Strains Cell Biology Recombinant Proteins Rats Dilution Solubility chemistry Electrophoresis Polyacrylamide Gel Dialysis Ammonium acetate |
| Zdroj: | Analytical Biochemistry. 176:326-329 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/0003-2697(89)90317-5 |
| Popis: | An efficient method has been developed for the purification of recombinant rat gamma-interferon (rat rIFN-gamma). The procedure involves extraction of the Escherichia coli cell paste with 6 M guanidine-HCl (GuHCl), adsorption of the rat rIFN-gamma onto C8 alkyl-bonded silica, and elution with 50% propanol. The protein is essentially pure at this step, but is quantitatively precipitated by threefold dilution with aqueous buffer at pH 8.5. The precipitate is then dissolved with 6 M GuHCl in a buffer containing 0.05%. Tween-80 to about 0.3 mg/ml and dialyzed against the same buffer. The rat rIFN-gamma, which remains soluble on dialysis is again precipitated by dialysis against ammonium sulfate at 80% saturation. This final precipitate is readily soluble in 0.1 M ammonium acetate buffer, pH 8.5. The preparation is fully active and possesses a specific activity of 2-6 X 10(6) units/mg. The recoveries ranged from 50 to 85% in several experiments. The sequence of 20 amino acid residues from the NH2-terminus of the protein was determined using an automated sequencer and was found to agree with that deduced from the cDNA sequence. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|