Lai C Y, Dharm E, Fujii Y
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110.
Anal Biochem. 1989 Feb 1;176(2):326-9. doi: 10.1016/0003-2697(89)90317-5.
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.
已开发出一种高效的重组大鼠γ干扰素（大鼠rIFN-γ）纯化方法。该程序包括用6M盐酸胍（GuHCl）提取大肠杆菌细胞糊，将大鼠rIFN-γ吸附到C8烷基键合硅胶上，并用50%丙醇洗脱。在此步骤中蛋白质基本纯净，但通过在pH 8.5的水性缓冲液中三倍稀释可定量沉淀。然后将沉淀物用含有0.05%吐温-80的6M GuHCl溶解至约0.3mg/ml，并在相同缓冲液中透析。透析时仍可溶的大鼠rIFN-γ通过在80%饱和度的硫酸铵中透析再次沉淀。该最终沉淀物易溶于pH 8.5的0.1M醋酸铵缓冲液中。该制剂具有完全活性，比活性为2-6×10(6)单位/mg。在几次实验中回收率为50%至85%。使用自动测序仪测定了该蛋白质NH2末端20个氨基酸残基的序列，发现与从cDNA序列推导的序列一致。
