Construction, Expression and Functional Characterization of the β-Lactamase with αv Integrin Ligands
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: &agrv Integrin Ligands; ACDCRGDCFCG peptide; ADEPT; ADEPT systems; Antibody-directed enzyme prodrug therapy; Chemotherapy; Cloning Vector; DNA Polymerase; DNA markers; FITC labeled RGD4CβL; HindIII; Imidazole; Lysozyme; MCF-7; NdeI; Ni-NTA resin; Plasmids; RGD4C; SDS-PAGE; T4 DNA ligase; Tricine-SDS-PAGE; Western blotting; agarose gel electrophoresis; agarose/Tris-acetate EDTA; aminopeptidase; breast cancer cell line; chloramphenicol; integrin αvβ3; lysis buffer; pColdII plasmid; recombinant DNA technology; recombinant protein; β-Lactamase
Document Type: Research Article
Publication date: 2010-12-01
- Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.