cDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein

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Abstract

Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad bard on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10-9 M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10-9 M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10-8 M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.

Original languageEnglish
Pages (from-to)5025-5032
Number of pages8
JournalJournal of Immunology
Volume150
Issue number11
Publication statusPublished - 1993
Externally publishedYes

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Chemokine CCL2
Recombinant Proteins
Organism Cloning
Guinea Pigs
Complementary DNA
Peritoneal Macrophages
Exudates and Transudates
Monocytes
Macrophages
High Pressure Liquid Chromatography
Amino Acids
Intradermal Injections
COS Cells
Sepharose
Open Reading Frames
Proteins
Spleen

ASJC Scopus subject areas

  • Immunology

Cite this

cDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein. / Yoshimura, Teizo.

In: Journal of Immunology, Vol. 150, No. 11, 1993, p. 5025-5032.

Research output: Contribution to journalArticle

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