In vitro cell free synthesis of human manganese superoxide dismutase with the RTS 500 system.
Reduced activity of manganese superoxide dismutase (MnSOD) is the basis of several pathologic features and complications occurring in the course of infectious mononucleosis. In order to supply future research with easily accessible enzyme, an in vitro protocol was developed based on the RTS 500 system and an overexpression vector. Translation of MnSOD monomers could be detected by SDS-PAGE, and assembly of the active homotetramer by native PAGE. Enzyme activity was successfully shown by in gel activity tests and enzyme assays. With 15 micro g of DNA, 2.45 micro kat were generated. The purification of MnSOD was performed by chromatography applying the His-tag technology. In SDS-PAGE of the eluate, a band showed up at M(r) 25000.
Kleines
Michael
M
Division of Virology, Department of Medical Microbiology, University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074, Aachen, Germany.
Nellessen
Martin
M
Schaade
Lars
L
Ritter
Klaus
K
D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D013482:Superoxide Dismutase / Q000096:biosynthesis*
D017931:DNA Primers; D013482:Superoxide Dismutase