RESUMO
OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.
Assuntos
Avidina/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Avidina/química , Avidina/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Biotina/análogos & derivados , Biotina/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/citologia , Escherichia coli/genética , Fluoresceínas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Confocal , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Trypanosoma cruzi undergoes a biphasic life cycle that consists of four alternate developmental stages. In vitro conditions to obtain a synchronic transformation and efficient rates of pure intermediate forms (IFs), which are indispensable for further biochemical, biological, and molecular studies, have not been reported. In the present study, we established an improved method to obtain IFs from secondary amastigogenesis. During the transformation kinetics, we observed progressive decreases in the size of the parasite body, undulating membrane and flagellum that were concomitant with nucleus remodeling and kinetoplast displacement. In addition, a gradual reduction in parasite movement and acquisition of the amastigote-specific Ssp4 antigen were observed. Therefore, our results showed that the in vitro conditions used obtained large quantities of highly synchronous and pure IFs that were clearly distinguished by morphometrical and molecular analyses. Obtaining these IFs represents the first step towards an understanding of the molecular mechanisms involved in amastigogenesis.