Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins.
In order for proteins to be used as pharmaceuticals, delivery technologies need to be developed 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) are used for the non-specific transduction of bio-active cargo, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intra-cellular protein that consists of 61 amino acids, including 20 cysteine residues, and is over-expressed under stressful conditions. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and by removing reactive oxygen species (ROS), and thereby prevent or reduce diabetes and diabetic complications, all MT applications have been made for gene therapy or under induced over-expression of endogenous MT. To overcome the drawbacks of ineffective intra-cellular MT protein uptake, a human MT gene was cloned and fused with protein transduction domains (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 three types of proteins were optimized by adding Zn ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn-MT cluster. The Zn-MT cluster showed better stability than MT in vitro. PTD-MT fusion proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemia-induced apoptosis with or without hypoxia. PTD-MT recombinant fusion proteins may be useful protein therapeutics for the treatment or prevention of diabetes and diabetes-related complications.
Lim
Kwang Suk
KS
Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seongdong-gu, Seoul 133-791, Republic of Korea.
Won
Young-Wook
YW
Park
Yong Soo
YS
Kim
Yong-Hee
YH
D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D008668:Metallothionein