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Protein expression and purification v.142, 2018년, pp.75 - 80   SCI SCIE
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Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization

Miranda, Beatriz Nogueira Messias (Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, 13566-590 São Carlos, SP, Brazil ) ; Fotoran, Wesley Luzetti (Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374 - Cidade Universitária, São Paulo, SP, Brazil ) ; Canduri, Fernanda (Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, 13566-590 São Carlos, SP, Brazil ) ; Souza, Dulce Helena Ferreira (Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, s/n, São Carlos, SP, Brazil ) ; Wunderlich, Gerhard (Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374 - Cidade Universitária, São Paulo, SP, Brazil ) ; Carrilho, Emanuel (Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, 13566-590 São Carlos, SP, Brazil ) ;
  • 초록  

    Abstract The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli . Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. Graphical abstract [DISPLAY OMISSION]


  • 주제어

    Alpha folate receptor .   Trigger factor .   Expression .   E.   coli .   Exosomes.  

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