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Abstract:
The human CC chemokine macrophage inflammatory protein-1α (MIP-1α) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1α gene had been fused to the Saccharomyces cerevisiae α-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1α was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1α revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1α was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1α was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1α receptor). Also, in competition binding assays the recombinant MIP-1α displayed high affinity binding.
