Publication Date

2016

Document Type

Dissertation/Thesis

First Advisor

Hofstetter, Oliver

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry

LCSH

Ligands (Biochemistry); Proteins

Abstract

Biorecognition is the ability of biological macromolecules, particularly proteins, to interact and form specific complexes with other macromolecules or small molecules, called ligands. Complementarity of the ligand to the binding site of the macromolecule with regard to both shape and electrostatic interactions increases the chances of the interaction to occur and stabilizes the resulting complex. The high specificity and affinity frequently observed with protein-ligand interactions has found wide application in fields such as biotechnology and medicine ranging from affinity separations to drug screening. In this work, specific protein-ligand interactions were exploited for two conceptually different types of applications. The first part of this dissertation describes the use of an anti-methotrexate single domain antibody for the separation of aminopterin, folate and methotrexate by immunoaffinity chromatography. In particular, the effect of random versus site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated. Genetic engineering techniques were utilized to modify the C-terminus of the antibody in order to facilitate its oriented coupling to a chromatographic support material and, thus, optimize the binding capacity of resulting columns. Frontal analysis was employed to compare two site-directed approaches with random antibody-coupling. The second part reports the utilization of biorecognition to target specific compounds in latent fingermarks. Since fingerprints are unique to an individual and do not change over time, they are considered to be the most important piece of physical evidence in criminal investigations. Here, three different types of biorecognition reagents, namely classically raised polyclonal antibodies, recombinantly expressed VHH antibodies, and lectins, were evaluated for their utility to visualize latent fingermarks based on their interactions with components contained in marks. Furthermore, a number of fusion proteins of VHHs and various fluorescent proteins were produced to allow for "direct" fingermark detection, i.e., visualization without the need of chemical labeling or additional visualization steps.

Comments

Advisors: Oliver Hofstetter.||Committee members: David Ballantine; Heike Hofstetter; Jim Horn; Joel Stafstrom.||Includes bibliographical references.||Includes illustrations.

Extent

xiii, 166 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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