AbstractThis work has been focused on the study of transport mechanisms of IPTG by E. coli in high cell density cultures.
Firstly, an HPLC-MS based method has been developed and validated under the FDA guidelines to quantify IPTG in both, medium and intracellular samples of fed-batch cultures for production of a recombinant aldolase. For the first time, the developed method was successfully applied to fed-batch sample and distribution profiles of IPTG were systematically assessed under different initial inducer concentrations.
Secondly, the results obtained from the study of IPTG distribution profiles allowed to discriminate between two different depletion patterns of inducer from medium to the biomass. Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Besides, we could study of induction behaviors of this expression system for recombinant protein production.
Then, the role of the lac-permease was studied and we could evidence that when lacking this transporter, diffusion of IPTG across the cell membrane can be assumed.
On the other hand, we extended the studies on IPTG transport to epoxide hydrolase producer system using an alternative operational strategy. The obtained results and the limited number of experiments did not allow us to compare with feasibility the results obtained for the RhuA system. However, we could also demonstrate that IPTG enters into the cell by active transport.
Finally, we used Response Surface Methodology aiming to optimize Rhamnulose-1-Phosphate aldolase (RhuA) production in fed-batch cultures. Previous optimization works within the research group did not consider the same variables. The effect of induction concentration, biomass concentration at induction time and specific growth rate were studied. Several responses were analyzed and we could obtain significant mathematical models that could predict maximum yields in terms of mg of protein. Validation experiments confirmed the predicted values obtained by the model. However, we did not achieve any successful model for RhuA activity since it is likely that other factors have a major influence and were not taken into account in this work.
To sum up, this thesis work has faced a controversial topic regarding the induction behavior in the recombinant protein production field. Our results were contrasted with these from the literature and intend to cover the lack of knowledge that was there to date. The obtained data expands the possibilities of research and it is expected that future works will be able to develop mathematic models capable to describe induction patterns together with maximization of recombinant protein yields.
|Date of Award||20 Apr 2012|
|Supervisor||Josep López Santin (Director) & Glòria Caminal Saperas (Director)|