Date of Graduation
Doctor of Philosophy in Engineering (PhD)
Robert R. Beitle
Edgar C. Clausen
Second Committee Member
Third Committee Member
Christa N. Hestekin
Fourth Committee Member
Applied sciences, Biotechnology, Metabolic flux analysis, Protein expression, Therapeutic proteins
Interest in the production of recombinant proteins consisting of collagen binding domain (CBD) fused to a bioactive material has increased due to the targeting/attachment capabilities of CBD. For example, CBD fusions can be applied to the reversing of bone density loss and the repair of the eardrum, specifically, by choosing an appropriate fusion partner (parathyroid hormone or epidermal growth factor). The production of CBD fusions was examined using batch and fed-batch culturing of Escherichia coli to express the fusion proteins, and affinity chromatography to isolate the final product.
Different medium formulations, feeding strategies, and induction methods were tested in order to develop a production strategy lacking yeast extract or other difficult-to-validate materials. Lactose was also examined as an alternative inducer to IPTG due to its lower cost and toxicity. This induction strategy, in conjunction with alternative feeding methods and the use of a completely defined medium, was able to produce the desired fusion proteins in a comparable manner to IPTG-induced systems. Also, the affinity tag on the N-terminus of the protein and the collagen binding domain on the C-terminus of the protein both retained their activity throughout the fermentation and purification processes.
The second portion of this dissertation examined and utilized two different types of models to mathematically describe the biological system. The first model was able to describe the fermentation system with respect to changes in feed, volume, biomass, and carbohydrate concentrations. This type of modeling examined the entire physical fermentation system on a 'macro' scale. Unlike the second model, it disregarded what occurred on a cellular level.
The second model utilized metabolic flux analysis to track changes in metabolite concentrations and biomass during the expression of the target protein. Upon solving this model, the prediction of the intermediate fluxes proved to be accurate for glucose-fed experiments, as the simulated carbohydrate concentrations match those that were experimentally determined. With the inclusion of the models, the work described in this dissertation provided a link between experimentally observed phenomena and mathematical descriptions of biological systems.
Fruchtl, M. S. (2013). Expression, Production, and Purification of Novel Therapeutic Proteins. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/701