DEVELOPMENT OF THE PRODUCTION AND PURIFICATION PROCESS OF THE GENETICALLY DETOXIFIED RECOMBINANT PNEUMOLYSIN OBTAINED IN ESCHERICHIA COLI
Abstract
Background
Current pneumococcal vaccines based on capsular polysaccharides are expensive and their coverage are limited to only few serotypes from >95 that are known, which led to a selective pressure that causes serotype replacement. Therefore, protein based vaccines have been investigated as an alternative and the virulence factor pneumolysin (Ply) is a promising candidate. This work aims at developing the production and purification process of the recombinant detoxified Ply (PdT), since reaching high recovery and purity of the antigen is essential for the development of affordable and safety vaccines.
Methods
E. coli + PdT cultivation was performed in 10L-bioreactor with auto-induction medium. After biomass suspensions, cells were lysed by chemical and physical methods. Different buffers were evaluated to enhance PdT solubility. After centrifugation, the supernatant was applied to chromatography steps. Protein concentration was measured by Lowry and BCA and PdT purity by densitometry of SDS-PAGE bands.
Results
The cultivation yielded 544 g wet biomass. The lysis and clarification processes promoted 94% recovery of soluble protein in bis-tris buffer pH 7.0. The chromatography steps allowed achieving < 50% PdT recovery.
Conclusions
Additional chromatography steps and process optimization will be evaluate in order to increase recovery, thus diminishing costs, and reach purity higher than 95%.
