High-level secretion of biologically active recombinant human macrophage inflammatory protein-1 alpha by the methylotrophic yeast Pichia pastoris

The human CC chemokine macrophage inflammatory protein-1 alpha (MTP-1 alpha ) was produced at a high level in Pichia pastoris under transcriptional con trol of the highly inducible alcohol oxidase 1 promoter. To ensure proper f olding and secretion of the recombinant polypeptide, the MIP-1 alpha gene h ad been fused to the Saccharomyces cerevisiae alpha-factor prepropeptide, A s was revealed by analysis of the cell culture supernatant of recombinant P ichia pastoris, MIP-1 alpha was efficiently secreted. Immunoblot analysis o f secreted proteins from recombinant clones using a polyclonal antibody dir ected against MIP-1 alpha revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1 alpha was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biolog ical activity of recombinant MIP-1 alpha was shown in a chemotaxis assay. H ere, the polypeptide specifically induced migration of U937 cells expressin g the CCR1 (MIP-1 alpha receptor). Also, in competition binding assays the recombinant MIP-1 alpha displayed high affinity binding. (C) 2000 Academic Press.