Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli

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Barazzone, Giovana C. ; Carvalho, Rimenys, Jr. ; Kraschowetz, Stefanie ; Horta, Antonio C. L. ; Sargo, Cintia R. ; Silva, Adilson J. ; Zangirolami, Teresa C. ; Goulart, Cibelly ; Leite, Luciana C. C. ; Tanizaki, Martha M. ; Goncalves, Viviane M. ; Cabrera-Crespo, Joaquin ; Spier, R

• Document type: Journal article
• Source: 4TH VACCINE AND ISV ANNUAL GLOBAL CONGRESS; v. 4, p. 9-pg., 2011-01-01.

Abstract

New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surface proteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensable for virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of protein sequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in 99% of strains associated with pneumococcal invasive disease. Hence, the purpose of this work was to develop an industrial production and purification process of His-tagged recombinant fragment of PspA in E. coli BL21 (DE3), rfPspA245 from family 1. Fed-batch cultivations in 5-L bioreactors with defined medium were carried out using glycerol as carbon source. It was obtained circa 60 g/L of dry cell weight and 3.0 g/L of rfPspA. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. The clarification step was done by centrifugation. The results of chromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographic sequence anion exchange (Q-Sepharose) followed by metal affinity (IMAC-Sepharose), the rfPspA245 was obtained with 67% and 97% of purity respectively for each step and final recovery of 23%. In conclusion, the purification process was developed and rfPspA245 was obtained with high purity, but the recovery should still be improved. (C) 2011 Published by Elsevier Ltd. Selection and peer-review under responsibility of Prof. Ray Spier (AU)

• FAPESP's process: 08/05207-4 - Pneumococcal conjugate vaccine: capsular polysaccharide - pneumococcal surface protein A
• Grantee: Martha Massako Tanizaki
• Support Opportunities: Research Projects - Thematic Grants