Biochemical and Biomolecular Engineering Commons^™

Membrane Chromatography For Bioseparations: Ligand Design And Optimization, Zizhao Liu

Graduate Theses and Dissertations

Membrane chromatography, or membrane adsorber, represents an attractive alternative to conventional packed bed chromatography used in downstream processing. Membrane chromatography has many advantages, including high productivity, low buffer consumption and ease to scale up. This doctoral dissertation focuses on developing novel polymeric ligands for protein separations using membrane chromatography. Atom transfer radical polymerization (ATRP), known as a controlled radical polymerization technique, has been used to control the architecture of grafted polymeric ligands. The center theme of this dissertation is to develop new polymeric ligands and investigate how the polymer’s property (e.g. flexibility, hydrophobicity) and architecture (e.g. chain density, chain length) …

Using Peptoids To Build Robust, Efficient Microarray Systems, Dhaval Sunil Shah

Graduate Theses and Dissertations

Recent studies have shown microarrays to be indispensable for various biological applications, allowing for high-throughput processing and screening of biological samples such as RNA, DNA, proteins and peptides using a small sample volume (< 1 µL). Peptoids (poly-N-substituted glycine oligomers) can be used as a substitute for antibodies as capture molecules, as well as coatings for slides in antibody microarrays. The ease of synthesis of peptoids, high customizability with desired bioactivity, and speed of synthesis allows us to build a diagnostic system with a large dynamic range that can detect biomolecules from a minimal sample size. In this study, peptoid-based antibody mimics are designed to have both structural and functional features similar to those of antibodies, including a stable constant region (scaffolding) and a variable region for protein recognition. Peptoids previously screened via combinatorial library synthesis to be specific to bind Mdm-2 (mouse double minute 2 homolog) and GST (gluthathione S-transferase), have been synthesized. The protein recognition peptoids have been conjugated to PEG (polyethylene glycol) molecules with modified end groups; an amine group on one end that allows for immobilization and orientation on the slide, and an azide group on the other end that will allow for attachment to the peptoid through “click chemistry”. The number of capture molecules printed on the slides can be increased by making the available surface area of the slide larger via coating with microspheres. We have determined that partially water soluble peptoids that are also helical, can self-assemble into microspheres. Sequences have been developed that can consistently produce uniform microsphere coatings on slides that increase the overall surface area. A high surface area corresponds to a higher number of binding sites, and therefore a more sensitive system. The work done has shown that slides may be successfully coated in order to potentially improve the detection system.

Expression, Production, And Purification Of Novel Therapeutic Proteins, Mckinzie Shea Fruchtl

Graduate Theses and Dissertations

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 …

Capillary And Microchip Electrophoresis For The Monitoring Of Disease Causing Amyloid Proteins, Elizabeth Nancy Pryor

Graduate Theses and Dissertations

The detection of oligomers and aggregates formed by two amyloid proteins, insulin and amyloid-beta (AB), is of particular importance due to the role which these species play in Diabetes and Alzheimer's disease, respectively. However, existing techniques are limited in the ability to detect insulin and AB; oligomers due to the fact that these early aggregates are transient, present at low concentrations, and difficult to isolate. Improvements must be made to existing techniques or alternative techniques must be explored in order to identify and quantify the size of these oligomeric and aggregate species without disrupting their structure.

Capillary and microchip electrophoresis …