Inyección directa de anticuerpos recombinantes monodominio al citoplasma de células humanas mediante los sst3 de e.Coli

• Autores: Ana Blanco
• Directores de la Tesis: Luis Ángel Fernández Herrero (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2010
• Idioma: español
• Tribunal Calificador de la Tesis: Manuel Fresno Escudero (presid.), Luis Álvarez Vallina (secret.), Junkal Garmendia (voc.), Antonio Juárez (voc.), Gad Frankel (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
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• Resumen

  • Many signaling and regulatory proteins found in the cytoplasm of human cells have a great potential as targets for therapeutic antibodies (Abs) but the plasma cell membrane prevents access to these antigens. Current technologies for cytoplasmic Ab expression in human cells require delivery of the Ab-encoding gene or fusion to protein transduction domains. Since Ab fragments and full IgGs can be selected and engineered in E. coli, this microorganism is an excellent candidate for their intracellular delivery. The work presented in this thesis demonstrates that single-domain Abs (sdAbs) can be engineered to be secreted and injected by E. coli into human cells using the molecular syringes assembled by a type III protein secretion system (T3SS) found in enteropathogenic and enterohemorragic E. coli strains (EPEC and EHEC). Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. To achieve this goal, a short 20 amino acid secretion signal from the natural T3 effector was fused to the N-terminus of sdAbs. The hybrid molecules were shown to be secreted to extracellular culture media by EPEC and EHEC strains carrying a functional T3SS. Secreted sdAbs were purified from culture supernatants and shown to be functional for specific antigen binding in vitro. Direct injection of sdAbs to the cytoplasm of HeLa cells was demonstrated by translocation assays using sdAb fusions to b-lactamase reporter and biochemical detection of sdAbs the cytoplasm of infected HeLa cells. Between 105-106 sdAb molecules accumulated per cell in the presence of antigen. Interestingly, expression of the antigen increases the level of sdAb detected in the cytoplasm of HeLa cells. Formation of the antigen-sdAb complex was demonstrated in the cytoplasm of HeLa cells. Finally, it is shown that attenuated EPEC strains with functional T3SS can be used for sdAb injection. Taken together, these results are proof-of-principle for the capacity of non-invasive E. coli bacteria to directly deliver functional sdAb polypeptides in the cytoplasm human cells.