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, homogenous 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.