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

被引:34
作者
PROOST, P
VANLEUVEN, P
WUYTS, A
EBBERINK, R
OPDENAKKER, G
VANDAMME, J
机构
[1] CATHOLIC UNIV LEUVEN,REGA INST,MOLEC IMMUNOL LAB,B-3000 LOUVAIN,BELGIUM
[2] APPL BIOSYST NEDERLAND,2800 AL GOUDA,NETHERLANDS
关键词
CHEMOKINE; CHEMOTAXIS; MONOCYTE; PROTEIN SYNTHESIS;
D O I
10.1006/cyto.1995.1013
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:97 / 104
页数:8
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