Simplified Method for Production and Purification of Tetanus
Tetanus toxin is produced by Clostridium tetani and is one of the most powerful toxins known. It has a molecular weight of approximately 150,000 and is produced within organisms and discharged into the medium as they autolyze.
Previously the toxin was prepared from culture filtrates after autolysis of the organisms. However, since Raynaud developed a method for extracting the toxin from organisms before they autolyzed (9), several groups have used bacterial extracts as starting material for purifying the toxin (1, 10). This method has the advantage that the toxin is obtained in a more concentrated and purer form than that from culture filtrates (9, 10).
In this study we used bacterial extracts as starting material and developed a simple, rapid method for purification of the toxin which included high–pressure liquid chromatography (HPLC). We first determined the optimal time for harvesting the organisms to extract the toxin reproducibly in maximal yield. For determining the optimal conditions for extraction, we studied the kinetics of release of the toxin from cells in hypertonic solution. Then we purified the toxin by techniques which included gel permeation chromatography by HPLC, a method which has recently become available and has the advantages of being rapid and giving high resolution.