PURIFICATION AND CHARACTERIZATION OF A NOVEL ENGINEERED MUTANT OF HUMAN α1-TYPE INTERFERON--IFN-α1/86D
Zhou Yuan Jin Dongyan Zhang Zhiqing Wang Wei Li Zhiliang Chi Jie Li Yuying Hou Yunde( National Laboratory of Molecular Biology and Genetic Engineering, Institute of Virology, Chtnese Academy of Preventive Medicine, Beijing 100052)
The kinetics of the expression of a novel engineered mutant of hu-man α1-type interferon ( IFN-α1/86D)in E.coli grown in shaken flasks or fermentor was studied. The target protein was then purified to95% homogeneity through cation exchange chromatograpy ( CM-Sepharose ) and affinity chromatography ( monoclonal antibody against IFN-α1 ) , the purified protein with a specific activity of 2.3×107 IU/mg protein, appeared as a single band on the SDS-PAGE electrophoretogram and a single peak on the HPLC chromatogram. The N-terminal amino acid se-quence of purified recombinant human IFN-α1/86D was determined on Applied Biosystems 477A protein/peptide sequencer and was shown to be correct. The purified protein was demonstrated to be heterogenous, co mprising of two sequences which differ merely by the N-terminal Met residue-25% Met-plusspecies and 75% Met-minus.