Recombinant and cellular expression of the murine chemotactic protein, CP-10.
The S100 protein CP-10 (chemotactic protein, 10 kD), a potent chemotactic factor for murine and human polymorphonuclear cells (PMN) and murine monocytes, has been purified in small amounts from supernatants of activated murine spleen cells (Lackmann et al., 1992). To obtain a more abundant source of the protein, CP-10 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The property of S100 proteins to undergo calcium-dependent conformational changes was used in a novel approach to optimize the release of recombinant (r) CP-10 by thrombin cleavage. Purified rCP-10 was characterized by amino-terminal sequence analysis and bioassays. Optimal chemotactic activity 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 protein bound to glutathione-agarose beads resulted in high titer, specific antibodies that neutralized CP-10-initiated chemotaxis and were suitable for immunoblotting. A combination of Western and Northern analyses identified CP-10 in murine peritoneal exudate PMN and macrophages, splenocytes, bone marrow cells, and WEHI-265 cells (all of myeloid origin), but not in thymus, liver, lung, 3T3 fibroblasts, EL4 lymphoma cells, or bEND 3 brain endothelial cells, indicating cell-specific regulation of CP-10 expression.
Iismaa
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Heart Research Institute Ltd, Camperdown, NSW, Australia.
Hu
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Kocher
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Lackmann
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Harrison
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Thliveris
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Geczy
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D040501:Calgranulin A; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000096:biosynthesis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D009418:S100 Proteins
D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D002630:Chemotactic Factors; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; C086145:S100a8 protein, mouse; D004247:DNA