Self-structured protein nanomaterials produced in endotoxin-free microbial cells
- Autores: Fabian Leonardo Rueda Alfonso
- Directores de la Tesis: Elena García i Fruitós (dir. tes.), Antonio Villaverde Corrales (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2016
- Idioma: inglés
- Tribunal Calificador de la Tesis: Paola Branduardi (presid.), Pau Ferrer Alegre (secret.), Antoni Benito Mundet (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Despite the enormous advances in recombinant technologies, Escherichia coli, the first-used biofactory, remains as the most used microorganism for the production of recombinant products. However, the lipopolysaccharide (LPS) contamination is an important bottleneck in the production of therapeutic proteins and/or any biomaterial coming from Gram-negative bacteria. Thus, the need to remove endotoxins is an important constraint for biomedical uses, considering that it does not exist a general purification method able to achieve a complete LPS clearance. In this context, the main objective of this thesis is to explore the use of LPS-free microbial cell biofactories for the production of different self-organized protein-based nanomaterials (inclusion bodies and cell targeted protein nanoparticles) and to determine their functional and structural features, as well as their potential applications in biomedicine. For that purpose, we have evaluated the potential of an endotoxin-free E. coli strain (KPM3335). Briefly, E. coli KPM335 have deletions in seven genes related to the biosynthesis of lipid A (LPS endotoxic domain), making E. coli KPM335 a safe cell factory for protein production purposes. In this context, VP1GFP functional inclusion bodies and T22GFPH6 tumor-targeted nanoparticles have been produced in the LPS-free E. coli strain with a similar yield than those evidenced in E. coli wild type strains. However, it is important to point that culture growth is slightly reduced in KPM335 strain and the produced nanoparticles show distinguishable features when compared to wild type strains. The different structural features and functional behavior of the LPS-free nanomaterials is attributed to the genetic modifications developed to eliminate endotoxicity. Thus, the involvement of the cell quality control in material’s architecture, cellular uptake and biodistribution of tumor-homing nanoparticles when administered in colorectal cancer animal models is discussed. In order to explain how the genetic background of different cell factories could directly impact on the functionality of recombinant nanomaterials at a macroscopic level, the tumor targeting model protein was also produced in E. coli strains lacking chaperones and proteases of the protein quality control system. In this regard, in vivo functional behavior of the tumor targeting nanoparticles produced in a strain lacking the DnaK protease was significantly different than those produced in either wild type or other mutant strains. Thus, the engineering of the protein quality control system of E. coli results in a number of conformational variants of protein nanoparticles with similar architectonic organization but different functional behavior. Either directly or indirectly, the introduction of mutations has an impact on the genetic background which could determine the structural organization and the efficiency of the nanoparticles biological activity.
On the other hand, other representative microbial cell factories such as Pichia pastoris were successfully used to produce functional VP1GFP inclusion bodies with similar architecture and biological activity than those produced in E. coli. While protein aggregates are commonly observed in recombinant bacteria and mammalian cells (as functional aggresomes), reports are missing about the formation of IBs or IB-like structures in yeast cells. Results obtained showed that VP1GFP IBs are able to maintain both architecture and functionality compared to those produced in standard cell factories, which makes it a robust choice independently of the biofactory selected.
In this regard, LPS-free biofactories proposed in this thesis can be used to produce safe cell-targeting nanoparticles as well as IBs for biomedical purposes with no LPS-associated risks. However, the genetic background is a relevant factor to be considered when designing production strategies of self-organized nanomaterials with special interest in biomedicine.
