Efficient production of active human manganese superoxide dismutase in Escherichia coli

Human manganese superoxide dismutase (hMnSOD) was expressed in Escherichia coli under regulation of the PL promoter and cII ribosomal binding site (rbs), derived from bacteriophage λ. Production of hMnSOD was induced upon temperature shift from 32°C to 42°C. The enzyme accumulated for a period of 120 min and reached levels about 25% of total bacterial proteins. Recombinant hMnSOD, of subunit molecular weight (MW) about 22 kDa, comigrated on NaDodSO4/polyacrylamide gels with authentic human liver MnSOD and reacted with antibodies directed against the latter. Mn++ supplementation in the bacterial growth media was essential for production of enzymatically active hMnSOD. The recombinant enzyme was purified to homogeneity by a combination of heat treatment and ion-exchange chro-matography. It behaves on gel filtration as a dimer of about 39 kDa and appears indistinguishable from the authentic enzyme, having a specific activity of 3500 units/mg, appropriate absorption spectrum and Mn content. Sequence analysis of the NH2-terminus, however, revealed an additional methionyl residue. The recovery of large quantities of pure hMnSOD with full enzymatic activity will permit in-depth evaluation of its clinical potential.