Purification and structural characterization of recombinant rat gamma-interferon from Escherichia coli.
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.
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Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110.
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Fujii
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D000595:Amino Acid Sequence; D000818:Animals; D011232:Chemical Precipitation; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D007371:Interferon-gamma / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins; D012995:Solubility
D011994:Recombinant Proteins; D007371:Interferon-gamma