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Background   Listeria monocytogenes  is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of  L. monocytogenes . In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work,  Lactococcus lactis  NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.    Results  The constructed vector (pNZPnisA:CYTO-LLO) was expressed in  L. lactis  NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30°C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.43–5.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4°C.    Conclusion  The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive  Lactococcus  bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in  L. lactis  has a number of benefits over  E. coli  which may facilitate both  in vivo  and  in vitro  applications of this system.

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000