High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein.
Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg 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. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.
Giammarini
Camilla
C
Centro di Biotecnologie, University of Urbino, Via T. Campanella 1, 61032 Fano, Italy.
Andreoni
Francesca
F
Amagliani
Giulia
G
Casiere
Annarita
A
Barocci
Simone
S
Magnani
Mauro
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001427:Bacterial Toxins; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006460:Hemolysin Proteins; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008089:Listeria monocytogenes / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C038705:hlyA protein, Listeria monocytogenes