High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli.

Abstract

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichwa coli by using a vector containing a thermoinducible X PL promoter and a ,8-lactamase-derived ribosomosal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented >10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD. and thus provide a defense against oxygen toxicity. There are three known forms of SOD that contain different metalsnamely, iron, manganese, or both copper and zinc. All of these catalyze the same reaction with high efficiency, and all operate by a similar mechanism in which the metal is the catalytic factor in the active site. These enzymes fall into several evolutionary groups. The Fe-containing SODs are found primarily in prokaryotic cells, while Cu/Zn SODs have been demonstrated in all higher eukaryotes. Mn SODs exist throughout the phylogenetic range, from microorganisms to humans (reviewed in ref. 4).Since every biological macromolecule can serve as a target for the damaging action of the abundant oxygen radical, interest has evolved in the therapeutic potential of SOD. A wide range of clinical applications has been suggested. These include prevention of oncogenesis and tumor promotion, reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (5), anti-inflammatory action (6), and protection against reperfusion damage of ischemic tissues (7). In addition, there is much interest in studying the effects of SOD on the aging process (8).The exploration of the therapeutic potential of human SOD has been hindered by its limited availability. The enzyme is a dimeric metalloprotein composed of identical noncovalently linked subunits, each of 16 kDa and containing one atom of copper and one atom of zinc (9). Each subunit is composed of 153 amino acids of known sequence (10, 11). Recently, a cDNA clone containing the entire coding region of human SOD was isolated and sequenced (12, 13). The gene coding for human SOD was introduced by us into an efficient bacterial expression vector. We report here the production of gram quantities of enzymatically active human Cu/Zn SOD in Escherichia coli. MATERIALS AND METHODSBacterial Gro...

Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri

“…This might not be the case for all other copper proteins, since, e.g., superoxide dismutase, having only a single copper atom per monomer (besides Zn), was shown to be functional when expressed in E. coli (20). Enzyme activity was found to depend on the copper concentration in the medium and required at least 0.39 mM Cu2+ (21). The gene for Alcaligenes faecalis S6 azurin also led to a functional product when expressed in E. coli (66).…”

Superoxide Dismutase from the Silkworm,Bombyx mori: Sequence, Distribution, and Overexpression

“…3,4,13) cDNAs encoding SODs from diverse organisms have been cloned, some of which were overexpressed in various hosts. [14][15][16][17][18][19][20][21] In spite of its important role in the regulation of ROS, the cDNA encoding a SOD of the silkworm is not yet available.…”

Amsacta mooreiEntomopoxvirus Expresses an Active Superoxide Dismutase

“…The k cat /K m value obtained indicates that AMVSOD is active, although 20-fold less active than bovine SOD at pH 8. The need for supplementation of the medium with copper and zinc for maximum activity has been demonstrated previously and hypothesized to be necessary, in part, due to the low levels of intracellular copper (20,28,45). We clearly saw multiple forms of the purified protein on Coomassie-stained gels, and the lower activity level of His-SOD may be due in part to the different forms of the protein that are present in the purified preparation, each of which may have different levels of activity.…”