Genetic and process strategies for the production of metallocarboxypeptidase inhibitors
- Autores: Juan Miguel Puertas Perez
- Directores de la Tesis: Gloria González (dir. tes.), Gloria Caminal Saperas (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2010
- Idioma: inglés
- Tribunal Calificador de la Tesis: Gena Larson (presid.), Montserrat Busquets Abió (secret.), Pau Ferrer Alegre (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
The central subject of the thesis is the development of efficient protocols to produce three carboxypeptidase inhibitors, rich in disulfide bridges, as secreted proteins in fed-batch cultures of recombinant E.coli strains. Different knowledge areas have been studied, mainly fermentation technology, expression vector design and protein secretion engineering.
In chapter 1, General introduction, an overview of the target molecules, recombinant protein production in E. coli and fermentation engineering is presented.
In chapter 2, Context and objectives, the aims of the present work are presented within the background from where they arise.
In chapter 3, Materials and methods, common protocols, reagents and media used throughout the work are described.
Chapter 4, Results, is composed by three pusblished research articles, which, toghether with the appended article of Chapter 5, Appendix, compile the results obtained during the thesis, as explained next: Initially, the work only focused on the production of the potato carboxypeptidase inhibitor (PCI). Therefore, Article I presents the design of a production process for this polypeptide, using the expression system MC1061 (pIMAM3) which had already been used in bioreactor cultures and rich media. A fed-batch protocol in minimal media was developed with the aims of directing most of the product to the extracellular culture broth. The influence of growth kinetics during the induction phase was studied, as well as the main bottlenecks affecting secretory protein yields. Also, an in-situ treatment was succesfully applied to release periplasmic-bound product into the culture media.
The main conclusion of this chapter was that the process was limited by the efficiency and operational stability of the pIMAM3 vector.
In order to improve production yields by improving plasmid stability and protein secretion, the construction of a new expression vector became necessary. For this purpose, leech carboxypeptidase inhibitor (LCI) was chosen as a model protein. This protein had been produced in shake flask cultures, but scale up to bioreactor had repeatedly failed. Article II, resulted from a six-month stay at the Unity of Structural Biochemistry of the Pasteur Institute in Paris, explains the development of an efficient Presentation 3 vector for the secretory expression of LCI. An arabinose-inducible system was chosen given the reduced cost of this sugar respect to IPTG; offering also the possibility of close control of expression. Several parameters were taking into account to maximize the amount of secreted product and the stability of the vector, which was named pLCB.
Article III is the result of another research stay, this time at the Chemical Engineering Department of the University of Washington, Seattle; carried out with the prupose of further increasing secretion of LCI with the pLCB vector. In it, two different genetic approaches were used to increase the levels of protein secretion. First, an E.coli strain carrying the deletion of the gene coding for the ribosomal protein Trigger Factor (TF) was used as expression background, which resulted in an increase on the levels of translocated inhibitor. Secondly, the main components the SRP machinery in prokaryotes, the RNA molecule ffs and the protein ffh, were coexpressed along with LCI, also with a positive influence.
From the promising results obtained in the works documented in Articles II and III, the genes coding for PCI and yet a new inhibitor isolated from the common tick Rhipicephalus bursa (TCI) were cloned in the KTD101/pLCB system with aims to develop a production process valid for these three proteins. In Article IV (still to be published) the recombinant strains were cultured in fed-batch with a similar protocol to that developed in Article I, resulting in significant yields of the three inhibitors and a robust process applicable to the production of a variety of secreted proteins in E.coli.
Also, comparison of the production processes allowed us to present some correlations between the effect of cysteine content and size and the expression and secretion of disulfide-bridged proteins.
A Summary of Results and General Discussion is given in chapter 6. Within, the results of the four articles are examined and analysed in order to highlight the main outcomes of these doctoral disertation and, at the same time, set a global overview of the results, listing its implications and applications.
Finally, in chapter 7 the main conclusions taken from the results and their anaylsis are presented.
