Development of the purification process of the genetically detoxified recombinant pneumolysin obtained in Escherichia coli

• Grant number: 18/13469-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2018
• End date: November 30, 2019
• Field of knowledge: Interdisciplinary Subjects
• Principal Investigator: Viviane Maimoni Gonçalves
• Grantee: Manuella Cazelato Pires
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Streptococcus pneumoniae is an important human pathogen that causes severe diseases with high mortality rates. Pneumococcal vaccines are based on capsular polysaccharides, but their production cost is high and their coverage is limited to the serotypes included in the formulation. Thus, the protein vaccines have been investigated as an alternative. Among the studied candidates highlights the virulence factor Pneumolysin (Ply) (Ply), a hemolytic toxin that binds to the plasmatic membrane, oligomerizes and generates pores that lyse host cells. Ply also binds to toll-like receptor 4, triggers complement activation by antibody-independent classic pathway, and recently it was shown that the pores generated activates the inflammasome NLRP3. Therefore, Ply have great vaccine and immunomodulatory potential, and due to its toxicity, it has been genetically detoxified for use in vaccines.The project aims at developing the purification process of the recombinant detoxified Ply(PdT), so that it can be evaluated in the future for a new pneumococcal formulation in nanoparticles for dry powder administration. PdT was cloned into Escherichia coli without any affinity tag to avoid the expensive and inefficient tag removal steps. The cultivation will be done in bioreactor with the animal-origin free auto-induction medium, according to the protocol already established for PdT production.For the purification process, the following steps will be evaluate: removal of fragments and impurities from the lysate, anion exchange chromatography and hydrophobic interaction chromatography, concentration/diafiltration as final conditioning step. Depending on the results obtained, if necessary, different strategies could be evaluated, such as: cation exchange, gel filtration or mixed interaction chromatography. Once established the purification and cultivation processes, they will be evaluated in a integrated manner in order to define the whole process with greatest recovery and purity.