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TOXICOLOGY

Expression and Purification of Glutathione Transferase-Small Ubiquitin-Related Modifier-Metallothionein Fusion Protein and Its Neuronal and Hepatic Protection against D-Galactose-Induced Oxidative Damage in Mouse Model

Biopharmaceutical Research and Development Center, Institute of Life and Health Engineering (Y.H., Z.S., Y.L., Q.Z., L.Cu., Y.S., C.D., M.Z., X.L.) and Center of Medicine and Biotechnology Research and Development (X.L.), Jinan University, Guangzhou, Guangdong, China; National Engineering Research Center of Genetic Medicine, Guangzhou, Guangdong, China (Y.H.); Shanxi Institute of Zoology, Xi'an, Shanxi, China (C.F.); Chinese-American Research Institute for Diabetic Complications, School of Pharmaceutical Sciences, Wenzhou Medical College, China (Y.T., W.F., X.L., L.Ca.); Department of Pediatrics, University of Louisville, Louisville, Kentucky (Y.T., W.F., L.Ca.); and Pharmaceutical College of Jinan University, Guangzhou, China (X.L.)

The present study aimed to produce and pathophysiologically evaluate the metallothionein (MT) fusion protein. A recombinant plasmid containing DNA segment coding the pET-glutathione transferase (GST)-small ubiquitin-related modifier (SUMO)-MT fusion protein was inserted into Escherichia coli for expression. The expression level of the fusion protein was very high, reaching to 38.4% of the total supernatant proteins from the organism. Subsequent filtration through glutathione Sepharose 4B gel and Sephadex G-25 yielded an MT fusion protein with purity more than 95%. When exposed to metals, E. coli containing the GST-SUMO-MT fusion protein showed an increased accumulation of Cd²⁺, Zn²⁺, or Cu²⁺ at approximately 4.2, 4.0, or 1.6 times higher, respectively, than those containing the control protein. Administration of GST-SUMO-MT to mice that were also treated with D-galactose to induce neuronal and hepatic damage showed a significant improvement of animal learning and memory capacity, which was depressed in mice treated by D-galactose alone. Administration of MT fusion protein also prevented D-galactose-increased malondialdehyde contents and histopathological changes in the brain and liver. Furthermore, supplement of the fusion protein significantly prevented D-galactose-increased nitric oxide contents and -decreased superoxide dismutase activity in the brain, liver, and serum. The fusion protein was also able to prevent ionizing radiation-induced DNA damage of the mouse thymus. The present study indicates that GST-SUMO-MT has a normal metal binding feature and also significantly protects the multiple tissues against oxidative damage in vivo caused by chronic exposure to D-galactose and by ionizing radiation. Therefore, GST-SUMO-MT may be a potential candidate to be developed for the clinical application.

Address correspondence to: Dr. Lu Cai, 570 South Preston Street, Suite 304F, Louisville, KY 40202. E-mail: l0cai001{at}louisville.edu