title: Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000.
abstract: 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 degrees 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 degrees C.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, University College Cork, Cork, Ireland. c.gahan@ucc.ie.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Bahey-El-Din
authors.forename: Brendan T
authors.initials: BT
authors.lastname: Griffin
authors.forename: Cormac Gm
authors.initials: CG
authors.lastname: Gahan