CHEMICAL SYNTHESIS, PURIFICATION AND FOLDING OF THE HUMAN MONOCYTE CHEMOTACTIC PROTEINS MCP-2 AND MCP-3 INTO BIOLOGICALLY-ACTIVE CHEMOKINES

Monocyte chemotactic proteins 2 and 3 (MCP-2 and MCP-3) are chemokines structurally and functionally related to MCP-1, In contrast to MCP-1, they are produced in low amounts by stimulated leukocytes or tumour cells, As an alternative method to generate sufficient protein for in vitro and in vivo characterization of MCP-2 and MCP-3, we have synthesized both 76-residue chemokines using Fmoc chemistry. After automated solid phase peptide synthesis at a 0.05 to 0.25 mmol scale, and purification to homogeneity by C-8 RP-HPLC, correct disulfide bridges were formed in a mixture of oxidized and reduced glutathione. The synthesis was biochemically controlled by peptide sequencing of intermediate products and proteolytic fragments of the 76-residue chemokines and by mass analysis, Purified synthetic MCP-2 and MCP-3 coeluted and comigrated with their natural counterparts on analytical reverse phase columns and SDS-PAGE, respectively, Purified and folded MCP-2 and MCP-3 were chemotactic for monocytes at 7.5 ng/ml and 5 ng/ml, respectively, These minimal effective concentrations are comparable to those of the natural chemokines, Synthetic MCPs did not induce neutrophil chemotaxis, Automated Fmoc peptide synthesis is thus a useful method, allowing fast production of chemokines and analogues thereof.