title: A chimera of green fluorescent protein with gelatinase binding and tumor targeting peptide.
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.
authors.affiliation: Department of Food and Environmental Sciences, Division of Microbiology, Viikinkaari 9, P.O. Box 56, University of Helsinki, FI-00014 Helsinki, Finland. per.saris@helsinki.fi
authors.forename: Justus
authors.initials: J
authors.lastname: Reunanen
authors.forename: Tanja-Maria
authors.initials: TM
authors.lastname: Ranta
authors.forename: Oula
authors.initials: O
authors.lastname: Peñate-Medina
authors.forename: Juho
authors.initials: J
authors.lastname: Suojanen
authors.forename: Timo
authors.initials: T
authors.lastname: Sorsa
authors.forename: Tuula
authors.initials: T
authors.lastname: Salo
authors.forename: Erkki
authors.initials: E
authors.lastname: Koivunen
authors.forename: Per E J
authors.initials: PE
authors.lastname: Saris
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007948:Leukemia, Monocytic, Acute / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C415426:CTTHWGFTLC peptide; D055767:Immobilized Proteins; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020780:Matrix Metalloproteinase 9; D000409:Alanine