Biochemical Studies on Superoxide Dismutase from Escherichia coli K12 and Deinococcus radiodurans R1
Date Issued
2005
Date
2005
Author(s)
Chin, Chia-Cheng
DOI
en-US
Abstract
The DNA fragment encoding manganese superoxide dismutase from
Deinococcus radiodurans and Escherichia coli was amplified and cloned into the
plasmid pQE30 to obtain an N-terminus His-tagged fusion expression plasmid. The
recombinant protein MnSODs were successfully overexpressed in E. coli M15
supplemented with Mn2+. The fusion proteins were purified in a single step by
Ni-NTA affinity chromatograph, and verified by western blotting and ESI-MASS
spectrometry. Characterization of oligomeric state by gel filtration chromatography
showed that three kinds of proteins were all dimeric. The specific activity of native E.
coli MnSOD, recombinant E. coli MnSOD, and recombinant D. radiodurans MnSOD
are 13000 U/mg, 9800 U/mg, and 7200 U/mg respectively.
Both recombinant MnSOD is shown to be more thermostable and radiation
resistance than native MnSOD of E. coli. The activity of native MnSOD reduced to
20% after heating at 80℃ for 20 min, whereas the activity of both recombinant
MnSODs was maintained at about 70% after heating at 90℃ for 20 min. His-tagged
MnSOD preserved more than 80% of its activity after irradiation, whereas native
MnSOD lost its activity dramatically.
Subjects
大腸桿菌
抗輻射菌
超氧岐化酶
熱穩定性
重組蛋白
E. coli
Deinococcus
superodxide dismutase
thermostability
recombinant protein
Type
other
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