Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications.
The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria. A protocol for large-scale expression and purification of recombinant LLO was previously optimized. By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media. Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.
Giammarini
Camilla
C
Centro di Biotecnologie, Università degli Studi di Urbino, Via T. Campanella 1, 61032 Fano, Italy.
Andreoni
Francesca
F
Amagliani
Giulia
G
Casiere
Annarita
A
Barocci
Simone
S
Magnani
Mauro
M
D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D002784:Cholesterol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D006460:Hemolysin Proteins; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D008089:Listeria monocytogenes / Q000276:immunology; D008088:Listeriosis / Q000175:diagnosis / Q000662:veterinary*; D008627:Mercuric Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis*; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D008627:Mercuric Chloride; D020245:p-Chloromercuribenzoic Acid; D002784:Cholesterol; C038705:hlyA protein, Listeria monocytogenes