In vitro expression of the restriction endonucleases LlaMI and ScrFI isolated from Lactococcus lactis M19 and UC503

A new restriction endonuclease LlaMI has been characterized in Lactococcus lactis subsp. cremoris M19. LlaMI recognizes the sequence 5′-CCNGG-3′ and cuts after the second cytosine. This restriction endonuclease is related to commercially available ScrFI but not identical to it. Comparative analysis of the predicted amino acid sequences of LlaMI and ScrFI indicates five non-conservative amino acid changes between these two restriction enzymes. These two enzymes were expressed in vitro as histidine-tagged fusion proteins. LlaMI was shown to be more sensitive to high salt concentration than ScrFI. Southern blotting and hybridization analysis indicate that the gene for LlaMI R/M system is chromosomally encoded.