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KMID : 0545120050150020330
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Journal of Microbiology and Biotechnology
2005 Volume.15 No. 2 p.330 ~ p.336
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Heterologous Expression of Human β-Defensin-1 in Bacteriocin-Producing Lactococcus Lactis
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Choi HJ
Seo MJ/Lee JC/Cheigh CI/Park H/Ahn C/Pyun YR
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Abstract
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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.
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