Pneumolysin (Ply) has been identified in all pathogenic pneumococcal isolates. This 53,000 MW protein is a thiol-activated cytolysin. It belongs to a class of bacterial cytolysins including: perfringolysin, listeriolysin and alveolysin which are characterized by their ability to lyse Sheep red blood cells (SRBC). It has been proposed that an inactivated pneumolysin could provide a protein carrier for a pneumococcal polysaccharide conjugate vaccine. The Type 19F pneumolysin gene was isolated from pneumococcal DNA utilizing oligonucleotides generated to the Type 2 pneumolysin gene in a polymerase chain reaction (PCR) resulted in a 1,420 bp fragment which was cloned into a pks(-) vector. The resulting clone p19F5 is positive for pneumolysin expression (SRBC assay and Western Blot) inside of E. coli DH5a. This clone represents our positive control to determine the level of toxin inactivation achieved with mutants. Truncating the protein prior to the single cysteine located near the carboxy terminus was the strategy selected to inactivate pneumolysin in our laboratory. PCR was utilized by generating an oligonucleotide which encodes a protein that terminates at the asparagine (ASN) located prior to the cysteine. The resulting 1,267 bp pneumolysin gene fragment was cloned into a pKK223-3 expression vector. The clone expressed a truncated pneumolysin protein of 47,386 MW which was not hemolytic. Purification of these proteins has proceeded using a parallel purification scheme to take advantage of the fact that 19F5 has hemolytic activity, which can be used to detect the protein throughout the purification strategy. The fractions corresponding to the SRBC lytic fractions assayed are pooled and passed over a Phenyl sepharose column equilibrated in sample buffer. The Ply fraction was detected by SRBC assay and was further purified using an HPLC Superose Column. Currently, we are scaling up our purification strategy to produce the quantities of purified protein, in milligrams, necessary for experimental conjugation.
