Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1.

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with lambda NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was expressed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded beta-casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plasmid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.