Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000

doi:10.1186/1475-2859-7-24

Microbial Cell Factories

Volume 7

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Research

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000

Mohammed Bahey-El-Din¹^,2 , Brendan T Griffin² and Cormac GM Gahan¹^,2^,3

¹Department of Microbiology, University College Cork, Cork, Ireland

²School of Pharmacy, University College Cork, Cork, Ireland

³Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

author email corresponding author email

Microbial Cell Factories 2008, 7:24doi:10.1186/1475-2859-7-24


Published:	29 July 2008

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°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.