RECOMBINANT AND CELLULAR EXPRESSION OF THE MURINE CHEMOTACTIC PROTEIN, CP-10

The S100 protein CP-10 (chemotactic protein, 10 kD), a potent chemotac tic factor for murine and human polymorphonuclear cells (PMN) and muri ne monocytes, has been purified in small amounts from supernatants of activated murine spleen cells (Lackmann et al., 1992). To obtain a mor e abundant source of the protein, CP-10 was expressed in Escherichia c oli as a fusion protein with glutathione S-transferase (GST). The prop erty of S100 proteins to undergo calcium-dependent conformational chan ges was used in a novel approach to optimize the release of recombinan t (r) CP-10 by thrombin-cleavage. Purified rCP-10 was characterized by amino-terminal sequence analysis and bioassays. Optimal chemotactic a ctivity of rCP-10 for murine PMN and WEHI-265 monocytoid cells was 10( -11) M (native protein has optimal chemotactic activity between 10(-11 ) and 10(-13) M). Immunization of rabbits with the GST/CP-10 fusion pr otein bound to glutathione-agarose beads resulted in high titer, speci fic antibodies that neutralized CP-10-initiated chemotaxis and were su itable for immunoblotting. A combination of Western and Northern analy ses identified CP-10 in murine peritoneal exudate PMN and macrophages, splenocytes, bone marrow cells, and WEHI-265 cells (all of myeloid or igin), but not in thymus, liver, lung, 3T3 fibroblasts, ELA lymphoma c ells, or bEND 3 brain endothelial cells, indicating cell-specific regu lation of CP-10 expression.