EXPRESSION OF A HUMAN MUTANT MONOCYTE CHEMOTACTIC PROTEIN-3 IN PICHIA-PASTORIS AND CHARACTERIZATION AS AN MCP-3 RECEPTOR ANTAGONIST

The cDNA encoding human monocyte chemotactic protein 3 (hMCP-3) was cl oned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris. After transfo rmation of P, pastoris by electroporation, several clones with the hum an MCP-3 gene integrated at the alcohol oxidase (AOX-1) locus were iso lated, One of these clones (M30) expressed the mature MCP-3 protein wi th three additional amino acids at its NH2 terminus as a secretion pro duct in the supernatant. The recombinant protein comigrated on SDS-PAG E and cross-reacted immunologically with synthetic hMCP-3, Intermediat e-scale production in shake flasks was obtained at expression levels o f approximately 1 mg per liter. The recombinant mutant MCP-3 was purif ied to homogeneity by adsorption on silicic acid, affinity chromatogra phy on heparin-Sepharose, and reversed-phase HPLC, At the amino termin us of the purified recombinant protein, the presence of the additional sequence Arg-Glu-Phe was confirmed by direct protein sequence analysi s, The recombinant hMCP-3 mutein was not glycosylated, as evidenced by deglycosylation experiments and by mass spectrometry, In analogy with MCP-1, the amino terminus of MCP-3 is crucial for its agonistic effec t on receptive cells, At concentrations up to 3.5 mu g/ml, the recombi nant mutein was not active in vitro as a chemotactic factor for monocy tes, However, the mutant MCP-3 acted as an MCP-3 receptor antagonist i n a competition chemotaxis assay at 100- to 1000-fold excess over the synthetic MCP-3 agonist. It might thus be a useful tool to study antag onism of MCP-3 action in vitro and in disease models of cancer and inf lammation.