Heterologous expression of human β-defensin-1 in bacteriocin-producing Lactococcus lactis

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human β-defensin-1 (hBD-1), which kills both Gram-positive and Gram-negative bacteria in L. lactis A164. When the hBD-1 cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-1 against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-1 gene was fused to the DsbC-Tag of pET-40b(+), then introduced to L. lactis A164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-1 fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-1, the DsbC-hBD-1 fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-1 exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A164 produced a biologically active hBD-1. In addition, the recombinant L. lactis A164 was also found to produce the same level of nisin Z as the wild-type.