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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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
Total Authors: 14
Document type: Journal article
Source: Applied Microbiology and Biotechnology; v. 101, n. 6, p. 2305-2317, MAR 2017.
Web of Science Citations: 3

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)

FAPESP's process: 12/04858-7 - Development of the purification process of the pneumococcal surface protein a from Clade4 (PspA4pro)
Grantee:Douglas Borges de Figueiredo
Support type: Scholarships in Brazil - Master
FAPESP's process: 15/06255-6 - Development and production of new pneumococcal vaccines based on recombinant proteins
Grantee:Viviane Maimoni Gonçalves
Support type: Regular Research Grants
FAPESP's process: 11/16605-3 - Cultivation of rE. coli in airlift bioreactor: development of strategies for optimization of biomass production
Grantee:Gilson Campani Junior
Support type: Scholarships in Brazil - Master