CLONING AND EXPRESSION OF RECOMBINANT HUMAN IMMUNE INTERFERON IN METHYLOTROPIC YEAST PICHIA PASTORIS AS A CONSTITUTIVE OR AS AN INDUCED PRODUCTION SYSTEM AND ITS USE THEREOF

摘要

The present invention describe a process for the extracellular production of a biologically active human interferon gamma protein from genetically modified methylotropic yeast Pichia species, preferably Pichia pastoris as constitutive production system,comprising of: The recombinant Pichia pastoris having at least one copy of constitutive expression cassette stably integrated in host genome, which is tandomly integrated by insertion events at GAP promoter locus of Pichia pastoris genome by a single crossover event between the said locus and Pgap region of the expression vector. This Pichia pastoris has at least one or more copies of such constitutive expression cassettes to produce high level of recombinant interferon gamma in fermented broth. This recombinant methylotropic yeast Pichia thus produces biologically active and properly folded interferon gamma polypeptide in fermented broth, in a constitutive manner when driven by the GAP promoter. A unique fermentation process was developed for this construct in Pichia host which produced interferon gamma. The yield was improved from ~ 8 mg/L to 40 mg/L. A method of partial purification process is also described.