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.

Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8+ T cells against Listeria monocytogenes in the murine infection model

“…2B). The above values of hemolytic activity are similar to those obtained for LLO expressed in other vectors [37,43].…”

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000

“…Although the LLO yield was good, those multi-step procedures are relatively time-consuming. To the best of our knowledge, Churchill et al were able to obtain the highest yield of haemolytically active LLO [ 14 ]. In their study, several expression systems have been attempted in E. coli among which the N-terminus His-tagged LLO was the most successful using the pQE31/IPTG (Isopropyl β-D-1-thiogalactopyranoside)-inducible system (Qiagen).…”

“…This system makes use of the well-characterized strong T5 promoter/ lac operator transcription-translation system to control LLO expression in E. coli . A significant LLO yield (3.5–8 mg per litre culture) was obtained with proven haemolytic activity (1.8 × 10 6 HU per mg protein) and stability up to one year upon storage at 4°C [ 14 ].…”

“…In the present work, we describe high level production of the L. monocytogenes LLO protein in Lactococcus lactis NZ9000 using the NICE system. The His-tagged LLO was purified by Ni-NTA affinity chromatography to give a considerable yield and high functional haemolytic activity compared to other published methods [ 14 , 15 ]. The significant yield and purity obtained for LLO using the NICE-expression system in Lactococcus demonstrate the benefits of this approach for the production of LLO for research and diagnostic purposes.…”

Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells

“…There have been several attempts to develop effective LLO purification methods using recombinant Escherichia coli strains [5-9]. Previously, we attempted to use modified bacteria species enclosed within capillary membranes using in vitro experiments with human T leukaemia Jurkat cells [10].…”