Abstract
Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5–8 mg l−1. The third contained a C-terminus His tag; it gave a yield of 2.5 mg l−1 LLO. The purified LLO from the latter two constructs retained its activity at 4°C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.
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Acknowledgements
Thanks to Yvan Chapdelaine of the National Research Council Canada for providing a vector containing the LLO gene. RLTC was the recipient of the Canadian Food Inspection Agency’s President’s Graduate Assistantship award. JCH was a recipient of the Canada Research Chair in Recombinant Antibody Technology. The authors were also funded by the Natural Sciences and Engineering Council of Canada and the Healthy Futures Program administered by the Ontario Ministry of Agriculture and Food.
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Churchill, R.L.T., Lee, H. & Hall, J.C. Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli . J IND MICROBIOL BIOTECHNOL 32, 355–363 (2005). https://doi.org/10.1007/s10295-005-0002-2
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DOI: https://doi.org/10.1007/s10295-005-0002-2