Metallothionein 융합단백질 개발과 이의 약제학적 응용에 관한 연구

Abstract

In order to proteins to be developed as biopharmaceutical drugs, various delivery technologies need to apply to overcome biochemical and anatomical barriers to protein drug transport, to protect proteins from systemic degradation, and to target the drug action to specific sites. Protein transduction domains (PTDs) have been used for the non-specific transduction of bio-active cargos, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intracellular protein that consists of 61 amino acids, including 20 cysteine residues, and over-expressed under stressful conditions such as heavy metals, starvation, or inflammation. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and removing reactive oxygen species (ROS), and thereby prevent or treat diabetes and diabetic complications, all MT studies have been focused on the therapeutic approach towards overexpression of endogenous MT. In the present study, a human MT gene was cloned and fused with PTDs, such as HIV-1 Tat and undeca-arginine, in a bacterial expression vector to produce PTD-MT fusion proteins. The expression and purification of PTD-MT fusion proteins were optimized by adding zinc (Zn) ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn-MT cluster. PTD-MT fusion proteins strongly protected Ins-1 beta cells and H9c2 cardiomyocytes against oxidative stress and apoptosis induced by ROS induced stress conditions. Intracellular uptake of Tat-MT was improved by short membrane transduction sequences (sMTS) derived from mitochondria malate dehydrogenase (mMDH) and measured their therapeutic effect in vitro. Functional activities of Tat-MT were also evaluated in streptozotozin (STZ)-induced diabetic mouse model and ischemic/reperfusion myocardial model. PTD-MT fusion protein maintained the blood glucose level at physiological levels in the STZ-induced diabetic mouse model and improved heart function in the ischemic/reperfusion myocardial model. The present data will promote future applications of MT recombinant fusion proteins for treatment and prevention of diabetes and diabetic complications.