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Doctoral Thesis
DOI
Document
Author
Full name
André Moreni Lopes
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Pessoa Junior, Adalberto (President)
Carvalho, João Carlos Monteiro de
Hasmann, Francislene Andreia
Kilikian, Beatriz Vahan
Mazzola, Priscila Gava
Title in Portuguese
Remoção de endotoxina presente em meio fermentado contendo biomoléculas utilizando sistemas micelares de duas fases aquosas
Keywords in Portuguese
Endotoxinas
GFPuv
Purificação de proteínas
Remoção de LPS
Triton X-114
Abstract in Portuguese
Foi investigada a utilização de Sistema Micelar de Duas Fases Aquosas (SMDFA) para remoção de lipolissacarídeos (LPS) de preparações contendo proteínas recombinantes de interesse farmacêutico, como a proteína verde fluorescente (GFPuv). Os SMDFA são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, que possibilitam seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, permitindo a remoção de LPS contaminante. Neste trabalho, foi realizada a implementação do método para a quantificação de LPS em amostras contaminadas e a obtenção de LPS e GFPuv puros a partir de cultivo de E. coli recombinante. Além disso, foi estudada a influência do Triton X-114 na metodologia de quantificação de LPS, e a adição de MgSO4, CaCl2, KI e (NH4)2SO4 na partição de GFPuv e LPS puros em SMDFA. E ainda, realizou-se um planejamento experimental (22) para avaliar os maiores KGFPuv e %RECGFPuv. O homogeneizado celular de E. coli foi testado nas melhores condições obtidas com o planejamento experimental. E finalmente, o processo por cromatografia de afinidade por íons metálicos (IMAC) foi empregado para investigar a adsorção de LPS em matriz IDA-Ca2+. Conforme os resultados obtidos, o TX-114 causou elevada interferência no método cinético cromogênico, em função da similaridade desta molécula com os LPS. Os LPS apresentaram partição preferencial para a fase concentrada em micelas, com altos valores de remoção, %REMLPS>98,0%. Ao contrário, a GFPuv foi recuperada preferencialmente na fase diluída, na qual existe maior volume disponível, resultando em valores de KGFPuv>1. A adição de sais ocasionou diminuição nos valores KGFPuv, provavelmente por causa da carga negativa que GFPuv adquiriu nas condições avaliadas. Os resultados do planejamento experimental mostraram que a melhor condição de partição obtida foi na região do ponto central, 4,0% (p/p) a 60,0°C, com KGFPuv>10. O processo por IMAC apresentou as maiores capacidades de adsorção de LPS-IDA-Ca+2 nas condições de menor pH e maior força iônica 4,0 e 1,0 mol/L, respectivamente. O processo de purificação por SMDFA empregado para a remoção de LPS contaminante presente em meio fermentado contendo GFPuv, demonstrou ser eficiente em recuperar a biomolécula-alvo na fase diluída e separar o principal contaminante na fase rica em micelas. Portanto, pode ser empregado como primeira etapa para a remoção de altas concentrações de LPS na purificação de proteínas hidrofílicas como a GFPuv.
Title in English
Endotoxin removal from fermentation broth containing biomolecules using two-phase micellar system
Keywords in English
Endotoxins
LPS removal
Protein purification and GFPuv
Triton X-114
Abstract in English
The Aqueous Two-Phase Micellar System (ATPMS) was investigated for endotoxin (LPS) removal from preparations containing recombinant proteins of pharmaceutical interest, such as the green fluorescent protein (GFPuv). These systems usually consist of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the biomolecules partitioning according to its hydrophobicity. In this work, the implementation of the method for LPS quantification in contaminated samples was accomplished, as well as the obtaining of pure LPS and GFPuv from recombinant E. coli. Furthermore, the influence of Triton X-114 in the methodology for LPS quantification was studied, as the addition of MgSO4, CaCl2, KI, and (NH4)2SO4 into the partition of pure GFPuv and LPS in ATPMS. In addition, a statistical design (22) was carried out to evaluate the highest KGFPuv and %RECGFPuv. The E. coli cell lysate was tested under optimum conditions obtained with the statistical design. And, finally, the process by ionmetal affinity chromatography (IMAC) was used to investigate the adsorption of LPS in IDA-Ca2+ matrix. The results showed that the TX-114 caused high interference in the kinetic chromogenic method, according to the similarity of this molecule to LPS. The LPS showed preferential partitioning to the micellerich phase, with high values of removal, %REMLPS>98.0%. In the other hand, the GFPuv was preferentially recovered in the micelle-poor phase, in which there is greater volume available resulting in values of KGFPuv>1. The addition of salts caused a reduction in the values KGFPuv, probably because of the negative charge that the GFPuv acquired at the conditions evaluated. The results of the statistical design showed that the best partitioning condition obtained was in the central point region, 4.0% (wt/wt) at 60.0°C, with KGFPuv>10. The process by IMAC showed the highest adsorption of LPS-IDA-Ca+2 capacities at the conditions of lower pH and higher ionic strength 4.0 and 1.0 mol/L, respectively. The purification process for the LPS contaminant removal from E. coli fermentation containing GFPuv by ATPMS proved to be efficient in the recovering of target biomolecule in the micelle-poor phase, and separating the main contaminant in the micelle-rich phase. Furthermore, this system can be exploited as the first step for removal of higher LPS concentrations from hydrophilics protein purification.
 
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AndreLopes.pdf (2.62 Mbytes)
Publishing Date
2011-04-04
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • JOZALA, A.F., et al. Liquid–liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems [doi:10.1016/j.enzmictec.2007.08.005]. Enzyme and Microbial Technology [online], 2008, vol. 42, n. 2, p. 107-112.
  • LOPES, André M., et al. Green fluorescent protein extraction and LPS removal from Escherichia coli fermentation medium using aqueous two-phase micellar system [doi:10.1016/j.seppur.2011.07.043]. Separation and Purification Technology [online], 2011, vol. 81, n. 3, p. 339-346.
  • LOPES, André M., et al. LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system [doi:10.1002/btpr.463]. Biotechnology Progress [online], 2010, vol. 26, n. 6, p. 1644-1653.
  • MAGALHAES, Pérola de Oliveira, et al. Methods of Endotoxin Removal from Biological Preparations: a Review. Journal of Pharmacy & Pharmaceutical Sciences, 2007, vol. 10, p. 271-287.
  • MAZZOLA, Priscila G, et al. Liquid–liquid extraction of biomolecules : an overview and update of the main techniques [doi:10.1002/jctb.1794]. Journal of Chemical Technology & Biotechnology [online], 2008, vol. 83, n. 2, p. 143-157.
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