A chimera of green fluorescent protein with gelatinase binding and tumor targeting peptide Journal Article
| Authors: | Reunanen, J.; Ranta, T. M.; Penate-Medina, O.; Suojanen, J.; Sorsa, T.; Salo, T.; Koivunen, E.; Saris, P. E. J. |
| Article 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 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. © 2010 Elsevier Inc. All rights reserved. |
| Keywords: | protein expression; genetics; metabolism; amino acid substitution; gelatinase b; green fluorescent protein; protein binding; cell line, tumor; chemistry; hybrid protein; recombinant fusion proteins; protein purification; escherichia coli; tumor cell line; green fluorescent proteins; alanine; peptides, cyclic; cyclopeptide; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; matrix metalloproteinases; affinity chromatography; gelatinases; ctthwgftlc peptide; immobilized protein; acute monocytic leukemia; chromatography, affinity; immobilized proteins; leukemia, monocytic, acute; matrix metalloproteinase 9 |
| Journal Title: | Protein Expression and Purification |
| Volume: | 72 |
| Issue: | 2 |
| ISSN: | 1046-5928 |
| Publisher: | Academic Press Inc., Elsevier Science |
| Date Published: | 2010-08-01 |
| Start Page: | 234 |
| End Page: | 237 |
| Language: | English |
| DOI: | 10.1016/j.pep.2010.03.023 |
| PUBMED: | 20371289 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Export Date: 20 April 2011" - "CODEN: PEXPE" - "Source: Scopus" |
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