Metallothionein 융합단백질과 Zinc 복합체의 안정화 조건확립에 관한 연구

Abstract

During the last decade, proteins and peptides as biopharmaceuticals have been successfully developed for efficient drugs. However, several limitations have to be solved including physical stability in aqueous solution, transduction efficiency, enzymatic susceptibility. To increase the protein stability through conformation structure, stability constants were quantitative base on metal-ligand, Zn-S. Metallothioneins (MTs) are intracellular metal-binding proteins including Zn, Cu, Cd, and Hg that highly conserved cysteine residues. These proteins have two separate domain followed by Zn3S9 and Zn4S11. The 7 Zn ions of metallothionein are in a tetrahedral coordination environment. The stability constant reveals value of Zn dissociation from metallothionein using Fluozon-3, fluorescent chelating agent. Metallothioneins regulate homeostasis of zinc that is capable of important cellular metabolism. When the redox state is changed in MT, it will influence a conformational stability. Therefore, concentration of released Zn can be quantitative to obtain the stability constant of metallothionein. Finally, this study can determine the protein expression conditions and storage conditions. Furthermore, to increase the protein stability through protein-polymer conjugation, metallothionein was synthesized with monomethoxypoly(ethylene glycol), non-toxic, non-immunogenicities, water-soluble synthetic polymer. PEGylation increases not only their solubility and circulation time due to increase of protein size but also effectively reduces immunogenicities. Effects of reaction pH, temperature and molar ratio of PEG to protein on synthesis were determined for optimum PEGylation yield by size-exclusion chromatography.