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.
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 Cd(2+), Zn(2+), or Cu(2+) 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.
Huang
Yadong
Y
Biopharmaceutical Research and Development Center, Institute of Life and Health Engineering, Jinan University, Guangzhou, Guangdong, China.
Su
Zhijian
Z
Li
Yanmei
Y
Zhang
Qihao
Q
Cui
Lejia
L
Su
Ye
Y
Ding
Changcai
C
Zhang
Minjing
M
Feng
Chengli
C
Tan
Yi
Y
Feng
Wenke
W
Li
Xiaokun
X
Cai
Lu
L
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001921:Brain / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005690:Galactose; D005982:Glutathione Transferase / Q000096:biosynthesis*; D006801:Humans; D008054:Lipid Peroxides / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D008668:Metallothionein / Q000096:biosynthesis*; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis*; D013482:Superoxide Dismutase / Q000378:metabolism
D000975:Antioxidants; D008054:Lipid Peroxides; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D009569:Nitric Oxide; D008315:Malondialdehyde; D008668:Metallothionein; D013482:Superoxide Dismutase; D005982:Glutathione Transferase; D005690:Galactose