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.
Proost
P
P
Rega Institute, University of Leuven, Belgium.
Van Leuven
P
P
Wuyts
A
A
Ebberink
R
R
Opdenakker
G
G
Van Damme
J
J
D000595:Amino Acid Sequence; D001681:Biological Assay; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D002630:Chemotactic Factors / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins; D009000:Monocytes / Q000187:drug effects / Q000502:physiology; D009504:Neutrophils / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding
C515450:CCL7 protein, human; C515453:CCL8 protein, human; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D002630:Chemotactic Factors; D016207:Cytokines; D004220:Disulfides; D018945:Monocyte Chemoattractant Proteins; D010446:Peptide Fragments; D010450:Endopeptidases