Production and purification of Streptococcus pneumoniae hemolysin (pneumolysin)

Pneumolysin was found to be produced by 112 of 113 clinical isolates of Streptococcus pneumoniae and to be an intracellular hemolysin. A 10-liter-scale fermentor production and purification procedure was developed for this hemolysin. The culture was concentrated by filtration 10 times before centrifugation. The cellular content was purified by ion-exchange chromatography, covalent thiopropyl gel chromatography, and gel filtration. One batch operation resulted in 6 mg of highly purified pneumolysin, with a yield of 66% and a specific activity of 1,400,000 hemolytic units per mg. The pneumolysin had a molecular weight of 53,000 and an isoelectric point of 5.2. The purification method developed will be of value in future studies on this hemolysin.