Immunoaffinity purification, stabilization and comparative characterization of listeriolysin O from Listeria monocytogenes serotypes 1/2a and 4b.
We developed a simple and highly effective procedure for stabilizing the haemolytic activity of listeriolysin O (LLO) from Listeria monocytogenes after immunoaffinity purification. The haemolytic activity of LLO was stabilized by eluting it directly into tubes containing an alkaline buffer (5 mM lysine, 140 mM KCl, 50% ethylene glycol, pH 11.5). The purified LLO retained 100% of its haemolytic activity after 6 weeks of storage at -20 degrees C. LLO purified from a strain of L. monocytogenes serotype 1/2a (ATCC 43249) and LLO purified from a strain of L. monocytogenes serotype 4b (F 2365) isolated from a Mexican-style cheese, showed no significant differences in pH and temperature stability. When incubated in buffers at pH values from 4 to 12 at 4 degrees C and 25 degrees C, LLO from serotypes 1/2a and 4b retained maximal haemolytic activity at pH 8 after 4 h of incubation. LLO from both serotypes lost their haemolytic activity after incubation at 50 degrees C for 25 min.
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Division of Bacterial and Mycotic Diseases, Centers for Disease Control, Atlanta, GA 30333.
Bibb
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WF
Helsel
L
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Dewitt
W
W
Swaminathan
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B
D001427:Bacterial Toxins; D002846:Chromatography, Affinity / Q000379:methods*; D004355:Drug Stability; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D066298:In Vitro Techniques; D008089:Listeria monocytogenes / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes