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

被引:8
作者
Barazzone, Giovana C. [1 ]
Carvalho, Rimenys, Jr. [1 ]
Kraschowetz, Stefanie [2 ]
Horta, Antonio C. L. [2 ]
Sargo, Cintia R. [2 ]
Silva, Adilson J. [2 ]
Zangirolami, Teresa C. [2 ]
Goulart, Cibelly [1 ]
Leite, Luciana C. C. [1 ]
Tanizaki, Martha M. [1 ]
Goncalves, Viviane M. [1 ]
Cabrera-Crespo, Joaquin [1 ]
机构
[1] Inst Butantan, Ctr Biotecnol, Av Vital Brazil 1500, BR-05503900 Sao Paulo, Brazil
[2] Univ Fed Sao Carlos, Dept Engn Quim, BR-13565905 Sao Carlos, SP, Brazil
来源
4TH VACCINE AND ISV ANNUAL GLOBAL CONGRESS | 2011年 / 4卷
基金
巴西圣保罗研究基金会;
关键词
PspA; Streptococcus pneumoniae; production; purification; HIGH-CELL-DENSITY; STREPTOCOCCUS-PNEUMONIAE; VACCINE; VIRULENCE; CARRIAGE; AGE;
D O I
10.1016/j.provac.2011.07.005
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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
引用
收藏
页码:27 / 35
页数:9
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