Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content

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Figueiredo, Douglas B. ; Carvalho, Eneas ; Santos, Mauricio P. ; Kraschowetz, Stefanie ; Zanardo, Rafaela T. ; Campani, Jr., Gilson ; Silva, Gabriel G. ; Sargo, Cintia R. ; Horta, Antonio Carlos L. ; Giordano, Roberto de C. ; Miyaji, Eliane N. ; Zangirolami, Teresa C. ; Cabrera-Crespo, Joaquin ; Goncalves, Viviane Maimoni

• Tipo de documento: Artigo Científico
• Fonte: Applied Microbiology and Biotechnology; v. 101, n. 6, p. 2305-2317, MAR 2017.
• Citações Web of Science: 3

Resumo

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 A degrees C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 +/- 2.5% of PspA4Pro with 97.8 +/- 0.36% purity and reduced endotoxin concentration by > 99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values. (AU)

• Processo FAPESP: 15/06255-6 - Desenvolvimento e produção de novas vacinas pneumocócicas baseadas em proteínas recombinantes
• Beneficiário: Viviane Maimoni Gonçalves
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 11/16605-3 - Cultivo de rE. coli em biorreator airlift: desenvolvimento de estratégias para otimização da produção de biomassa
• Beneficiário: Gilson Campani Junior
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 12/04858-7 - Desenvolvimento do processo de purificação da proteína a de superfície do pneumococo do clado 4 (PspA4Pro)
• Beneficiário: Douglas Borges de Figueiredo
• Modalidade de apoio: Bolsas no Brasil - Mestrado