Construction, expression and functional characterization of the β-lactamase with αv integrin ligands.
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 α(v)β(3) 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, β-Lactamase moiety 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.
Wang
Hao
H
Institute of Radiation Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, 300192 Tianjin, China.
Shi
Pei-Ji
PJ
Wu
Ming-Fen
MF
Li
Ning
N
Zhou
Xiao-Liang
XL
Fan
Fei-Yue
FY
D016503:Drug Delivery Systems; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008024:Ligands; D009842:Oligopeptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D039302:Integrin alphaVbeta3; D008024:Ligands; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases