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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.

In recent years, the infusion of therapeutic proteins (i.e. insulin, growth factors, cytokines, etc.) into the medical field has opened new avenues for researchers to explore treatments for many of the ailments that currently accost society. Attesting to their importance and enormous potential is the fact that therapeutic proteins have become the fastest growing area of pharmaceuticals. Despite the ascribed benefits of these biopharmaceuticals, following administration, they are often recognized as foreign by the body and consequently removed from circulation, preventing them from carrying out their designed function for prolonged periods of time. The work presented herein examines the glycosylation of biopharmaceutical proteins as means of improving their therapeutic effectiveness. Specifically, this work describes two novel approaches for the site-selective and indiscriminate glycosylation of proteins, procedures that are unique in the fact that they allow the conjugation of high molecular weight carbohydrates—up to 10,000 Daltons and beyond—to the surface of proteins. To highlight the usefulness of the developed methods, three biopharmaceuticals were selected for glycosylation and their effects subsequently studied. The developed methodology was initially applied to allow for the chemical glycosylation of hemoglobin, a protein widely researched for its potential to serve as a blood substitute in transfusion medicine. In summary, hemoglobin was successfully site-selectively glycosylated and the impact of glycosylation on its various biophysical properties subsequently investigated. The second protein selected for chemical glycosylation was the catalytic enzyme paraoxonase-1 (PON1), a protein valued for its potential to provide prophylactic protection from exposure to a multitude of highly toxic organophosphate compounds—many of which are currently stockpiled around the world for deployment in chemical warfare. Following indiscriminate glycosylation, the enzyme’s catalytic efficiency was investigated and its potential to serve as a viable prophylactic treatment subsequently studied. Lastly, the methods developed for the site-selective glycosylation of hemoglobin and the indiscriminate glycosylation of PON1 were employed for the modification of human serum albumin (HSA)—a protein currently used as a plasma expander in transfusion medicine. Additionally, described herein is a procedure for the synthesis of glycopolymers capable of self-assembling to form glyconanoparticles in aqueous environments at physiological temperatures. In summary, the GNPs were designed and synthesized to serve as anti-adhesion agents capable of removing Shiga toxin (Stx)—the lethal toxin produced by Escherichia Coli—from the body.In a separate area of research discussed herein, glycosyltransferases—the superfamily of enzymes responsible for the construction of complex carbohydrates and glycoconjugates—were investigated for their usefulness as powerful synthetic tools. Specifically, detailed in this work is a novel method for analyzing the efficiency and ability of glycosyltransferases to accept non-natural substrates for the synthesis of carbohydrate derivatives. To demonstrate the applicability of the described methodology, a panel of 1,2-fucosyltransferases was investigated for their ability to synthesize the H(O) blood group trisaccharide and related analogs from a variety of chemoenzymatically synthesized GDP-fucose derivatives. Lastly, reported in this work is an endeavor undertaken to better understand the reversibility of glycosylation reactions catalyzed by glycosyltransferases.

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application.