Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli
Barazzone, Giovana C; Carvalho, Rimenys Jr; Kraschowetz, Stefanie; Horta, Antonio C. 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.
Procedia in Vaccinology
; 4: 27-35, 2011.
Artigo
Inglês
| SES-SP, SES SP - Instituto Butantan, SES-SP, SES SP - Acervo Instituto Butantan | ID: biblio-1065735
New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surfaceproteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensablefor virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of proteinsequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in99% 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. Itwas 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 ofchromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographicsequence 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.
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BR78.1
Localização: BR78.1
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