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

作者：Reunanen Justus; Ranta Tanja Maria; Penate Medina Oula; Suojanen Juho; Sorsa Timo; Salo Tuula; Koivunen Erkki; Saris Per E J^*

来源：Protein Expression and Purification, 2010, 72(2): 234-237.

DOI：10.1016/j.pep.2010.03.023

• Protein expression
• Protein purification
• Green fluorescent protein
• Matrix metalloproteinases
• Gelatinases

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 4 beta-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.

• 出版日期2010-8