High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Giammarini, Camilla; Andreoni, Francesca; Amagliani, Giulia; Casiere, Annarita; Barocci, Simone; Magnani, Mauro

doi:10.1016/s1046-5928(02)00682-4

Cited by 15 publications

(30 citation statements)

References 17 publications

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“…These extra amino acids were not anticipated to have any effect on the activity of His-LLO. Furthermore, Giammarini et al [9] showed that the removal of the secretion sequence does not affect the activity of LLO.…”

Section: Analysis Of the Construct Sequencesmentioning

confidence: 99%

“…Giammarini et al [9,10] increased the expression of a truncated LLO that lacked the secretion sequence by using an E. coli expression system, then purified the LLO on a hydroxyapatite column, concentrated it by ammonium sulfate precipitation, and further purified it using SP Sepharose cation-exchange chromatography. The authors were able to increase the recovery of LLO to 4.5 mg l À1 [9], but the columns used and requirement for SDS-PAGE gels after each chromatography step and hemolysis assays to confirm the presence of the protein added time and complexity to the procedure.…”

Section: Expression and Purification Of Llo Via The Pqe Vectorsmentioning

confidence: 99%

“…However, these methods are time consuming, requiring days for purification, and result in very low yields of LLO, taking as much as 10 l culture to purify 132 lg LLO [29], or at best, 3 l culture for 4.8 mg protein [4]. More recently, some researchers have purified truncated recombinant LLO, lacking the secretion signal, from Escherichia coli [9,10]. Although the authors were able to produce 4.5 mg l À1 LLO, their methods were labour intensive and time consuming, requiring adsorption to hydroxyapatite, ammonium sulfate precipitation, and cation exchange chromatography [9].…”

Section: Introductionmentioning

confidence: 99%

“…More recently, some researchers have purified truncated recombinant LLO, lacking the secretion signal, from Escherichia coli [9,10]. Although the authors were able to produce 4.5 mg l À1 LLO, their methods were labour intensive and time consuming, requiring adsorption to hydroxyapatite, ammonium sulfate precipitation, and cation exchange chromatography [9].…”

Section: Introductionmentioning

confidence: 99%

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Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Churchill¹,

Lee²,

Hall³

2005

J IND MICROBIOL BIOTECHNOL

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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.

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Section: Analysis Of the Construct Sequencesmentioning

confidence: 99%

Section: Expression and Purification Of Llo Via The Pqe Vectorsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Churchill¹,

Lee²,

Hall³

2005

J IND MICROBIOL BIOTECHNOL

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“…This strain may also be applied as a source of pure LLO because L. lactis produces neither inclusion bodies nor endotoxins, which differs from recombinant Escherichia coli expressing LLO containing lipopolysaccharide in its outer membrane (Giammarini et al, 2003). Finally, we are currently evaluating the DNA transfer capacity of this novel L. lactis strain in experiments performed with intestinal epithelial cells in vitro.…”

mentioning

confidence: 99%

Prospective uses of recombinant Lactococcus lactis expressing both listeriolysin O and mutated internalin A from Listeria monocytogenes as a tool for DNA vaccination

Azevedo¹,

Rocha²,

Pereira³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. In this study, Lactococcus lactis was engineered to express mutated internalin A on its surface and to secrete large amounts of listeriolysin O (LLO) in order to improve its potential as a vehicle for DNA vaccination. Western blotting experiments demonstrated that the bacterium expressed LLO in both the cytoplasmic and extracellular compartments, with higher quantities found in the culture supernatants. A hemolytic assay showed that the recombinant strain secreted 250 ng active LLO/mg total protein. This mInlA/LLO-producing strain of L. lactis may be used as an alternative tool in DNA vaccination against a number of infectious diseases or in cancer therapy.

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Listeriolysin O as cytotoxic component of an immunotoxin

2009

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Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine-oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell-type-unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell-type-specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor-antigen Lewis Y. The coupling of LLO to dsFv-B3 was performed via cysteine-containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3-LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC 50 value of 2.3 nM, whereas antigen negative cell lines were 80-to 250-fold less sensitive towards B3-LLO.

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High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Cited by 15 publications

References 17 publications

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Prospective uses of recombinant Lactococcus lactis expressing both listeriolysin O and mutated internalin A from Listeria monocytogenes as a tool for DNA vaccination

Listeriolysin O as cytotoxic component of an immunotoxin

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