Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification.
Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.
Darji
A
A
Department of Cell Biology and Immunology, GBF, Braunschweig, Germany.
Chakraborty
T
T
Niebuhr
K
K
Tsonis
N
N
Wehland
J
J
Weiss
S
S
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001427:Bacterial Toxins; D001709:Biotechnology; D003001:Cloning, Molecular; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008087:Listeria / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D011817:Rabbits; D014774:Virulence / Q000235:genetics
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes