[Food-grade expression of human Cu/Zn-superoxide dismutase gene in Lactococcus lactis]

A food-grade gene expression system in L. lactis using the lacF gene as selection marker was constructed and further used for food-grade expression of human Cu/Zn superoxide dismutase (Cu/Zn SOD). Firstly, an integrative plasmid pUCEmDE containing homologous fragments with 0.5 kb flank sequences of the lacF gene was constructed. The lacF gene was in-frame deleted by double cross-over between the plasmid pUCEmDE and the chromosomal DNA in L. lactis MG5267 and resulted in a food-grade host WZ103 that was confirmed by PCR and Lac phenotype examination. After that, a complementary plasmid pMG36eF in which the lacF gene was controlled by the strong constitutive promoter P32 was electroporated into WZ103 and resulted in the restoration of Lac+ phenotype, indicating that the lacF function in WZ103 could be complemented by the lacF gene in pMG36eF. Finally, a food-grade plasmid pWZ104 used for the expression of Cu/Zn SOD was constructed, in which the lacF gene was used as a selective marker instead of any antibiotic resistance genes. Expressed Cu/Zn SOD in WZ103 (pWZ104) was demonstrated and showed biological activity through non-denatured PAGE and SOD activity gel-staining.