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Mn-superoxide dismutase (MnSOD) had been cloned from rice
cDNA library. PCR was used to insert the DNA (mmnsod) coding for
the rice mature MnSOD protein into the pGEX-4T-1 expression
vector. The recombinant DNA was transformed to Escherichia coli
BL21 and the construct was identified by enzyme digestion,
southern, PCR and DNA sequence analysis. Expression of GST-MnSOD
fusion protein was induced by adding 0.1 mM IPTG to bacterial
cultures and the homogeneous GST-MnSOD was purified by the GST-
glutathione affinity system. But most of GST-MnSOD protein in E.
coli resulted in an insoluble aggregate which could be recovered
by 6M urea and dialysis. Thrombin treatment could cleave GST-
MnSOD into GST and rMnSOD(recombinant MnSOD, which had extra
five amino acids in N terminal sequence than rice instric
MnSOD). Both purified GST-MnSOD and rMnSOD had SOD activity, and
were still insensitive to KCN and H2O2, which were
characteristics of MnSOD. 10 % SDS polyacrylamide gel showed
that the monomer molecular weight of the two proteins was 50 kDa
and 23 kDa. The native form, estimated by 5-20 % gradient
polyacrylamide gel electrophoresis, was dimer both in GST-MnSOD
and rMnSOD. The isoelectric point of rMnSOD was 4.64, but GST-
MnSOD was in the range of pH 4.74 - pH 4.97. The SOD activity
declined to 30 % when the two protein incubated at 60 ℃ for 20
minutes, but more stable at alkaline pH environment. In vitro
transcription containing T7 RNA polymerase and [a-35S] CTP are
used to estimate the RNA relative products. The results
indicated that GST (glutathione-S-transferase) can dramatically
enhance the transcription, but SOD showed no this function.
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