P. Proost et al., CHEMICAL SYNTHESIS, PURIFICATION AND FOLDING OF THE HUMAN MONOCYTE CHEMOTACTIC PROTEINS MCP-2 AND MCP-3 INTO BIOLOGICALLY-ACTIVE CHEMOKINES, Cytokine, 7(2), 1995, pp. 97-104
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 c
ells, As an alternative method to generate sufficient protein for in v
itro and in vivo characterization of MCP-2 and MCP-3, we have synthesi
zed both 76-residue chemokines using Fmoc chemistry. After automated s
olid phase peptide synthesis at a 0.05 to 0.25 mmol scale, and purific
ation to homogeneity by C-8 RP-HPLC, correct disulfide bridges were fo
rmed in a mixture of oxidized and reduced glutathione. The synthesis w
as biochemically controlled by peptide sequencing of intermediate prod
ucts and proteolytic fragments of the 76-residue chemokines and by mas
s analysis, Purified synthetic MCP-2 and MCP-3 coeluted and comigrated
with their natural counterparts on analytical reverse phase columns a
nd SDS-PAGE, respectively, Purified and folded MCP-2 and MCP-3 were ch
emotactic for monocytes at 7.5 ng/ml and 5 ng/ml, respectively, These
minimal effective concentrations are comparable to those of the natura
l chemokines, Synthetic MCPs did not induce neutrophil chemotaxis, Aut
omated Fmoc peptide synthesis is thus a useful method, allowing fast p
roduction of chemokines and analogues thereof.