Preparation and Characterization of Recombinant Tissue Inhibitor of Metalloproteinase 4 (TIMP-4)

• Published in: The Journal of biological chemistry Vol. 272; no. 33; pp. 20479 - 20483
• Main Authors: Liu, Yiliang E., Wang, Mingsheng, Greene, John, Su, Jeffery, Ullrich, Stephen, Li, Hui, Sheng, Shijie, Alexander, Preston, Sang, Qingxiang Amy, Shi, Y. Eric
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.08.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Breast Neoplasms - pathology; Breast Neoplasms - therapy; Female; Glycoproteins - biosynthesis; Glycoproteins - isolation & purification; Glycoproteins - pharmacology; Humans; Neoplasm Invasiveness; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Tissue Inhibitor of Metalloproteinases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.33.20479

TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Rosen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375–30380). In this report, the production and characterization of recombinant TIMP-4 (rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected insect cells, was purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatographies. The purified protein migrated as a single 23-kDa band in SDS-polyacrylamide gel electrophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p was demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 values (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nm for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degradation and may have therapeutic potential in treating cancer malignant progression.