ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Filtering by
- All Subjects: Biochemistry
- Creators: Hecht, Sidney
drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the
cost, labor and time associated with production of antibodies focus has recently changed
towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy
to work with, non-immunogenic, cost effective and amenable to scale up. Even though
researchers have developed several affinity peptides, we are far from compiling library of
peptides that encompasses entire human proteome. My thesis describes high throughput
pipeline that can be used to develop and characterize affinity peptides that bind several
discrete sites on target proteins.
Chapter 2 describes optimization of cell-free protein expression using commercially
available translation systems and well-known leader sequences. Presence of internal
ribosome entry site upstream of coding region allows maximal expression in HeLa cell
lysate whereas translation enhancing elements are best suited for expression in rabbit
reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate
combination ensures maximum protein expression of DNA libraries.
Chapter 3 describes mRNA display selection methodology for developing affinity peptides
for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant
is not sufficient to increase selection pressure for up to three rounds of selection and
increasing number of selection rounds increases probability of finding affinity peptide s.
These studies enhance fundamental understanding of mRNA display and pave the way
for future optimizations to accelerate convergence of in vitro selections.
Chapter 4 describes a high throughput double membrane dot blot system to rapidly
screen, identify and characterize affinity peptides obtained from selection output. I used
dot blot to screen potential affinity peptides from large diversity of previously
ii
uncharacterized mRNA display selection output. Further characterization of potential
peptides allowed determination of several high affinity peptides from having Kd range 150-
450 nM. Double membrane dot blot is automation amenable, easy and affordable solution
for analyzing selection output and characterizing peptides without ne ed for much
instrumentation.
Together these projects serve as guideline for evolution of cost effective high throughput
pipeline for identification and characterization of affinity peptides.
The second part of Chapter 1 is concerned with ribosomal protein synthesis in bacteria. Ribosome, the organelle that synthesizes proteins with exceptional fidelity, has a strong bias for α-L-amino acids. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome could enable the incorporation of both α-D-amino acids and β-amino acids into full length protein.
Oxidative stress is a common cause of various neurological disorders such as Alzheimer’s disease and Parkinson’s disease. Antioxidative strategies are used widely for the treatment of these disorders. Although several antioxidants demonstrated positive results in vitro as well as in in vivo models, none of them have been effective in clinical settings. Hence, there is an ongoing search for effective neuroprotective drugs. Described in Chapter 2 is the synthesis and biological evaluation of several methylene blue analogues as potentially effective antioxidants for the treatment of pathologies related to oxidative stress.
In Chapter 3, the synthesis and ribosomal incorporation of several rationally designed dipeptidomimetic analogues are discussed. The dipeptidomimetic analogues are structurally similar to the GFP chromophore and, therefore, highly fluorescent. In addition, the backbone of the dipeptidomimetic analogues resemble the peptide backbone of a dipeptide, due to which they can be incorporated into protein by modified ribosomes selected for the incorporation of dipeptides.
Discussed in Chapter 4 is the synthesis of the pdCpA derivatives of several β-amino acids. The pdCpA derivatives were ligated to tRNA-COH and were used as probes for studying the regio- and stereoselectivity of modified ribosomes.