Abstract
Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents at high concentration to tumor tissues while minimizing systemic drug exposure. β-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity, which allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. This study used integrin αv β3 as a target for tumor-specific delivery of β- Lactamase. β-Lactamase was fused with ACDCRGDCFCG peptide (RGD4C) by recombinant DNA technology. Likewise, this study cloned a fused cDNA and successfully expressed active recombinant protein in E. coli purified with Ni- NTA resin. After purification, the protein showed the expected size of 42 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein was found to be active for specificity in breast cancer cell line, MCF-7, which supports the utility of the protein as an agent for ADEPT.
Keywords: ADEPT, β-Lactamase, RGD4C, recombinant protein, Integrin Ligands, Antibody-directed enzyme prodrug therapy, ADEPT systems, integrin αvβ3, ACDCRGDCFCG peptide, recombinant DNA technology, Ni-NTA resin, Tricine-SDS-PAGE, Western blotting, breast cancer cell line, MCF-7, Chemotherapy, aminopeptidase, Plasmids, DNA Polymerase, T4 DNA ligase, DNA markers, Cloning Vector, agarose/Tris-acetate EDTA, pColdII plasmid, NdeI, HindIII, agarose gel electrophoresis, chloramphenicol, SDS-PAGE, lysis buffer, Lysozyme, Imidazole, FITC labeled RGD4CβL
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