Study of binding and fluorescence resonance energy transfer between the ECFP-labeled neocarzinostatin protein carrier and rhodamine 123

• Main Authors: Yao-Ping Wang, 王耀平
• Other Authors: Der-Hang Chin
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/j746qs

碩士 === 國立中興大學 === 化學系所 === 100 === Neocarzinostatin is a potent antitumor antibiotic from Streptomyces carzinostaticus. It consists of a carrier apoprotein and an enediyne chromophore, which is known to be responsible for the cytotoxicity of neocarzinostatin. The apo-neocarzinostatin protects the labile chromophore and exhibits high stability against a variety of denaturants. These features make the apo-neocarzinostatin a superior drug carrier for many nonenediyne molecules. Here, we aimed to establish a fluorescence resonance energy transfer pair using the apo-neocarzinostatin carrier and a protein-bound fluorophore. Such a protein-ligand binding pair may allow one to follow in real-time the process of drug release in cellular environment by employing florescent imaging techniques. In this study, we found that the fluorophore rhodamine 123 was able to bind tightly with apo-neocarzinostatin. The binding constant was independently examined by two different methods: fluorescence quenching titration and isothermal titration calorimetry. By protein engineering approaches, we fused an enhanced cyan fluorescent protein with the apo-neocarzinostatin. When rhodamine 123 was treated with the fusion protein, we successfully observed a fluorescence resonance energy transfer. In this protein-ligand pair, the fusion protein served as a fluorescence donor, whereas the apo-neocarzinostatin bound rhodamine 123 was a fluorescence acceptor. Our results provided a solid case for a fluorescence resonance energy transfer pair between a protein carrier and a ligand. Such a device can be a potential candidate to develop a new drug-delivery vehicle for therapeutic applications.