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Master's Dissertation
DOI
10.11606/D.87.2018.tde-22102018-152147
Document
Author
Full name
Douglas Borges de Figueiredo
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Gonçalves, Viviane Maimoni (President)
Azzoni, Adriano Rodrigues
Barazzone, Giovana Cappio
Title in Portuguese
Desenvolvimento do processo de purificação da proteína A de superfície de pneumococo do clado 4 (PspA4Pro).
Keywords in Portuguese
Streptococcus pneumoniae
Brometo de cetiltrimetilamônio
Cromatografia líquida preparativa
PspA
Purificação de proteínas
Abstract in Portuguese
A proteína A de superfície de pneumococo (PspA) é encontrada na superfície de todas as cepas de Streptococcus pneumoniae e candidata promissora para novas vacinas pneumocócicas. Foi desenvolvido um processo de purificação da PspA4Pro cujas etapas iniciais foram: ruptura da biomassa celular, precipitação do homogenato obtido com o detergente brometo de cetiltrimetilamônio (CTAB) e remoção do precipitado por centrifugação. Foram avaliadas cromatografias de troca iônica (aniônica, catiônica), afinidade por metais, interação hidrofóbica e mista de troca catiônica e hidrofóbica. Utilizando precipitação com CTAB, cromatografia de troca aniônica, crioprecipitação em pH4,0 e cromatografia de troca catiônica atingiu-se a pureza requerida de PspA4Pro (>95%) com recuperação entre 14% e 33%. O processo alcançou níveis aceitáveis de endotoxina no produto final e a PspA4Pro purificada foi reconhecida por anticorpos anti-PspA4, manteve sua atividade e sua estrutura secundária.
Title in English
Development of the purification process of pneumococcal surface protein A clade 4 (PspA4Pro).
Keywords in English
Streptococcus pneumoniae
Cetyltrimethylammonium bromid
Pneumococcal surface protein A
Preparative liquid chromatography
Protein purification
Abstract in English
Pneumococcal surface protein A (PspA) is found in all Streptococcus pneumoniae strains and is a promising candidate to be used in new pneumococcal vaccines. A purification process for PspA4Pro which inicial steps were: cell disruption, precipitation of the homogenate with the cationic detergent cetyltrimethylammonium bromide (CTAB) and pellet removal by centrifugation. The chromatographic techniques tested were ion exchange (anionic and cationic), immobilized metal affinity, hydrophobic interaction and mix mode with hydrophobic and cationic ligands. Using CTAB precipitation, anion exchange chromatography, crioprecipitation in pH4.0 and cation exchange chromatography the PspA reached the required purity (>95%) with recovery between 14% and 33% . The process reached acceptable levels of endotoxin in the final product and the purified PspA4Pro was recognized by anti-PspA4 antibodies and manteined its activity and secondary structure.
 
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Publishing Date
2018-10-22
 
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