Produção e purificação de um fragmento recombinante da proteína A de superfície do clado 3 (PspA3) de Streptococcus pneumoniae em Escherichia coli.

Abstract

CARVALHO, R. Jr. Production and purification of a recombinant fragment of pneumococcal surface protein A clade 3 (PspA3) from Streptococcus pneumoniae in Escherichia coli. 2009. Master thesis (Biotechnology) – Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, 2009. New vaccines against Streptococcus pneumoniae have been developed, among them a new conjugated vaccine using as carriers pneumococcal surface proteins has been studied. The protein pneumococcal surface protein A (PspA) was the first choice as carrier because it is indispensable for virulence of S. pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment of PspA clade 3 (rfPspA3) in E. coli BL21(DE3). Kinetic assays were performed, and then fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. For the clarification step, besides centrifugation, it was tested cross flow micro and ultra filtrations in hollow fibers, but these methods led to rfPspA3 loss (58% and 29%, respectively). Therefore, centrifugation was defined for the clarification step. Two chromatographic steps and the best sequence of them were analyzed: anion exchange (Q-Sepharose) and metal affinity (IMAC-Sepharose). The sequence with Q followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). For the third chromatographic step, three methods were evaluated: hydrophobic interaction (Phenyl-Sepharose), gel filtration (Sephacryl S200 HR) and cation exchange (SP-Sepharose). The last one was chosen for the purification process. The results of rfPspA3 purification from cultures using glucose or glycerol were compared and no relevant differences were found in the Q-Sepharose step. The final purity of rfPspA3 was 90%, but the recovery of the last chromatography was lower than previously obtained. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).