Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18

Many of the species of Lactobacillus can be considered to be "probiotics" with a variety of benefits, including imparting antioxidative effects to the host. Lactobacillus species evolve different mechanisms to defend themselves against oxygen toxicity, such as superoxide dismutases (SODs), hydroperoxidases and high intracellular levels of metal ions. The SODs provide a cellular defense mechanism against oxidative stress by scavenging O-2(-). Most Lactobacillus species appear to lack a sod gene. To date, only two species of Lactobacillus (Lactobacillus casei and L. sakei) may have sod genes, as evidenced by sequence analysis of the genome, but no experimental verification, including cloning and heterologous expression of the Lactobacillus sod gene, has been reported. It is therefore unknown whether these sod genes can express SOD or are functional. We have PCR-amplified the gene from L. casei Lc18 that encodes the SOD using primers designed according to the genome of L. casei ATCC 334 and ligated it into the vector pET-28a(+) for heterologous expression in Escherichia coli BL21(DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The superoxide radical scavenging activity of the recombinant strain was found to be increased relative to that of the control strain. The SOD was also purified by nickel ion affinity chromatography and found to consist of a single band, as determined by SDS-PAGE analysis, with an activity of 39.97 U/mg. N-terminal amino acid sequence analysis indicated that it may be a manganese-containing SOD. This is the first report of a sod gene from Lactobacillus spp. being expressed in other prokaryotes.