A chimera of green fluorescent protein with gelatinase binding and tumor targeting peptide

• Published in: Protein expression and purification 2010-08, Vol.72 (2), p.234-237
• Main Authors: Reunanen, Justus, Ranta, Tanja-Maria, Peñate-Medina, Oula, Suojanen, Juho, Sorsa, Timo, Salo, Tuula, Koivunen, Erkki, Saris, Per E.J.
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Amino Acid Substitution; Cell Line, Tumor; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gelatinases; Green fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Immobilized Proteins - chemistry; Immobilized Proteins - genetics; Immobilized Proteins - metabolism; Leukemia, Monocytic, Acute - metabolism; Matrix Metalloproteinase 9 - metabolism; Matrix metalloproteinases; Peptides, Cyclic - chemistry; Peptides, Cyclic - genetics; Peptides, Cyclic - metabolism; Protein Binding; Protein expression; Protein purification; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.03.023

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 4β-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.