title: Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli.
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 degrees 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.
authors.affiliation: Department of Environmental Biology, University of Guelph, N1G 2W1 Guelph, ON, Canada. jchall@uoguelph.ca
authors.forename: Robin L T
authors.initials: RL
authors.lastname: Churchill
authors.forename: Hung
authors.initials: H
authors.lastname: Lee
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification*; D006460:Hemolysin Proteins; D006461:Hemolysis / Q000187:drug effects; D008089:Listeria monocytogenes / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C038705:hlyA protein, Listeria monocytogenes