High-yield expression in Escherichia coli of soluble human MT2A with native functions.
Metallothioneins (MTs) are a family of low molecular weight, cysteine rich heavy metal binding proteins with multifunction, such as metal detoxification and antioxidation, and are involved in a number of cellular processes including gene expression, apoptosis, proliferation and differentiation. However, high yield expression of human MT in Escherichia coli has not been established effectively. To produce large amounts of human MT protein at low cost, recombinant human metallothionein 2A (MT2A) protein with an N-terminal GST tag was successfully expressed at high levels in soluble form in E. coli and high purification of it was established by affinity chromatography under native conditions. The final yield was about 5mg of the recombinant MT2A per liter of bacterial culture with the purity of 97.9%. Chemical and functional characteristics analysis of the recombinant human MT2A exhibited intact metal binding ability, hydroxyl radical scavenging ability and significant protective role against DNA damage caused by UVC radiation. Establishment of highly purified recombinant human MT2A protein with native characteristics at low cost would improve its function study and wide applications in protecting against oxidative damage and UV radiation.
Yang
Fang
F
Cancer Research Institute, Xiangya School of Medicine, Central South University, 90# Xiangya Road, Changsha, Hunan 410078, PR China.
Zhou
Min
M
He
Zhimin
Z
Liu
Xiaorong
X
Sun
Lin
L
Sun
Yu
Y
Chen
Zhuchu
Z
D002478:Cells, Cultured; D002846:Chromatography, Affinity; D020552:Comet Assay; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008214:Lymphocytes / Q000528:radiation effects; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008670:Metals / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D013054:Spectrophotometry, Atomic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays
C491053:MT2A protein, human; D008670:Metals; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D008668:Metallothionein; D005978:Glutathione