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

• Published in: Procedia in vaccinology Vol. 4; pp. 27 - 35
• Main Authors: Barazzone, Giovana C., Carvalho, Rimenys, Kraschowetz, Stefanie, Horta, Antonio L., Sargo, Cíntia R., Silva, Adilson J., Zangirolami, Teresa C., Goulart, Cibelly, Leite, Luciana C.C., Tanizaki, Martha M., Gonçalves, Viviane M., Cabrera-Crespo, Joaquin
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 2011
• Subjects: production; PspA; purification; Streptococcus pneumoniae; PspA; production; purification; Streptococcus pneumoniae
• ISSN: 1877-282X; 1877-282X
• DOI: 10.1016/j.provac.2011.07.005

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 60g/L of dry cell weight and 3.0g/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.