Abstract |
Matrix metalloproteinases ( MMPs) are enzymes that can hydrolyze almost all constituents of extracellular matrix. An MMP subgroup, the gelatinases, has been focused during last years, since over-expression of gelatinase A (MMP-2) and gelatinase B (MMP-9) has been linked with severe homeostasis disorders such as tumor growth, metastasis formation, and chronic inflammation. In this study, a phage display library-derived novel antigelatinolytic decapeptide, the CTT- peptide, was expressed as a carboxyl terminal, histidine-tagged fusion with the green fluorescent protein (CTT-GFP) in Escherichia coli. In addition, a biologically intact chimera, in which residues in the CTT- peptide critical for gelatinase binding were replaced with alanine (Ala-CTT-GFP), was constructed. The GFP-fusion proteins were purified to homogeneity with a simple one-step procedure utilizing nickel affinity chromatography. The purified chimeras were tested for their binding properties to 4beta-phorbol-12,13-butyrate (PdBu) activated, MMP-9 expressing THP-1 cells, and it was demonstrated that the CTT-GFP strongly bound to the cells, whereas Ala-CTT-GFP lacked the binding ability. Furthermore, the adherence of the CTT-GFP to MMP-9 expressing cells was demonstrated to be mediated by the CTT-moiety, since the binding could be dose-relatedly inhibited with increasing concentrations of synthetic soluble CTT- peptide. In conclusion, this novel tool, combining the gelatinase binding ability of the CTT- peptide with the fluorescing property of the GFP, should clearly improve both experimental and clinical studies of the role and function of gelatinases.
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Authors | Justus Reunanen, Tanja-Maria Ranta, Oula Peñate-Medina, Juho Suojanen, Timo Sorsa, Tuula Salo, Erkki Koivunen, Per E J Saris |
Journal | Protein expression and purification
(Protein Expr Purif)
Vol. 72
Issue 2
Pg. 234-7
(Aug 2010)
ISSN: 1096-0279 [Electronic] United States |
PMID | 20371289
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Crown Copyright 2010. Published by Elsevier Inc. All rights reserved. |
Chemical References |
- CTTHWGFTLC peptide
- Immobilized Proteins
- Peptides, Cyclic
- Recombinant Fusion Proteins
- Green Fluorescent Proteins
- Matrix Metalloproteinase 9
- Alanine
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Topics |
- Alanine
(metabolism)
- Amino Acid Substitution
- Cell Line, Tumor
- Chromatography, Affinity
- Electrophoresis, Polyacrylamide Gel
- Escherichia coli
- Green Fluorescent Proteins
(chemistry, genetics, metabolism)
- Humans
- Immobilized Proteins
(chemistry, genetics, metabolism)
- Leukemia, Monocytic, Acute
(metabolism)
- Matrix Metalloproteinase 9
(metabolism)
- Peptides, Cyclic
(chemistry, genetics, metabolism)
- Protein Binding
- Recombinant Fusion Proteins
(chemistry, genetics, metabolism)
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