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 cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris. After transformation of P. pastoris by electroporation, several clones with the human MCP-3 gene integrated at the alcohol oxidase (AOX-1) locus were isolated. One of these clones (M30) expressed the mature MCP-3 protein with three additional amino acids at its NH2 terminus as a secretion product in the supernatant. The recombinant protein comigrated on SDS-PAGE and cross-reacted immunologically with synthetic hMCP-3. Intermediate-scale production in shake flasks was obtained at expression levels of approximately 1 mg per liter. The recombinant mutant MCP-3 was purified to homogeneity by adsorption on silicic acid, affinity chromatography on heparin-Sepharose, and reversed-phase HPLC. At the amino terminus of the purified recombinant protein, the presence of the additional sequence Arg-Glu-Phe was confirmed by direct protein sequence analysis. 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 effect on receptive cells. At concentrations up to 3.5 micrograms/ml, the recombinant mutein was not active in vitro as a chemotactic factor for monocytes. However, the mutant MCP-3 acted as an MCP-3 receptor antagonist in 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 antagonism of MCP-3 action in vitro and in disease models of cancer and inflammation.
Masure
S
S
Laboratory of Molecular Immunology, Rega Institute for Medical Research, Leuven, Belgium.
Paemen
L
L
Proost
P
P
Van Damme
J
J
Opdenakker
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D054410:Chemokine CCL7; D003001:Cloning, Molecular; D016207:Cytokines; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D010843:Pichia; D018121:Receptors, Cytokine / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000096:biosynthesis
C515450:CCL7 protein, human; D054410:Chemokine CCL7; D016207:Cytokines; D018076:DNA, Complementary; D018945:Monocyte Chemoattractant Proteins; D018121:Receptors, Cytokine; D011994:Recombinant Proteins