Publication Date

2008

Document Type

Dissertation/Thesis

First Advisor

Hofstetter, Oliver

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry

LCSH

Enantiomers; Molecular biology; Biochemistry

Abstract

It is long known that living organisms can respond differently to the enantiomers of chiral molecules. While one enantiomer of, for example, a drug may cause a desired pharmacological effect, the other may cause undesired side-effects or even be harmful. Regulatory agencies such as the FDA, therefore, demand that the enantiomeric composition of all chiral drugs be determined. While host molecules commonly used for direct enantiomer analysis are typically found on a trial and error basis, it has been previously demonstrated that antibodies can serve as tailor-made chiral selectors. The Hofstetter group recently produced highly stereoselective anti-amino acid antibodies by classical immunological techniques. These antibodies were employed in a variety of analytical techniques for enantiomer detection and separation. The objective of this work was to utilize molecular biological techniques to obtain antibody fragments that stereoselectively recognize amino acids. Two different approaches were followed to produce scFv and Fab fragments, respectively, in bacterial cell cultures. In the first approach, a phage display library was produced utilizing the genetic material of a mouse that had been immunized with an immunogen carrying the hapten L-phenylalanine. Several phages displaying anti-L-amino acid-specific scFv were isolated and utilized for the expression of soluble scFv in E. coli. Characterization by ELISA demonstrated that the antibody fragments were highly stereoselective. In the second approach, a stereoselective Fab fragment was constructed using the genetic material of a hybridoma clone that produces an anti-L-amino acid antibody. Primers were designed to enable amplification of the V L, CL, VH, and CH1 sequences by PCR. Overlap PCR was used to obtain the sequences encoding the Fd and LC fragments, respectively, which were separately expressed in E. coli. Assembly of functional Fab was achieved by combining the two proteins under refolding conditions. A high degree of stereoselectivity was observed in both noncompetitive and competitive ELISA experiments.

Comments

Includes bibliographical references (pages 135-165)

Extent

xvi, 174 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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