Both technological and experimental advancements in the field of biotechnology have allowed scientists to make leaps in areas such nucleic acid, antibody, and recombinant protein technologies. Here we focus on the use of recombinant proteins as molecular recognition motifs, wound healing biomaterials, and agents for cell cycle pathway elucidation are discussed.
The author's primary project is described in chapters 2 and 3, and is focused on designed leucine-rich repeat proteins which offer increased stability, modularity, and surface area for binding interactions. These proteins bind at least two muramyl dipeptide ligands with picomolar to nanomolar affinity (Kd1 = 0.04 – 3.5 nM); as measured by fluorescence quenching experiments and ITC. The longest designed repeat, CLRR8, has a Kd app value of 1.0 nM which is comparable to full length native NOD2 protein. Molecular docking simulations revealed the locations of two potential binding sites and their respective interactions. The series of proteins represents a foundation for a high affinity and highly specific molecular recognition scaffold that has the potential to bind a variety of ligands.
Previously the author contributed to the design of recombinant keratin proteins, and the work in Chapter 4 builds on the original design to allow for controlled degradation in wound healing systems. Site-directed mutagenesis was utilized to introduce these degradation sites, and modified keratin proteins were expressed with no differences to native recombinant keratin proteins. Success in engineering a variation of native keratin protein with no issues in expression lay the foundation for further engineering of native keratin or other relevant proteins for improved functionality.
Chapter 5 describes steps towards producing human Aurora borealis (Bora) protein, an important substrate in cell cycle regulation, by in vitro transcription-translation with locked Ser–Pro analogues. This will allow for the elucidation of the active isomerization form to ensure proper cell division. Site-directed mutagenesis successfully introduced the amber codon to relevant Ser-Pro sites at positions 274 and 278. These mutated Bora genes along with modified ribosomes and aminoacyl tRNA will allow for the incorporation of locked dipeptide analogues. Expression of native Bora was carried out as a control, and appeared to express in dimeric form. The experiments carried out in Chapter 5 describe and outline all the molecular biology work completed and to be completed for this novel method of studying cis-trans isomerization in living cells. / Doctor of Philosophy / Sequencing of the human genome and the rapid development of gene editing and recombinant DNA technologies paved the way for a massive shift in the pharmaceutical industry. The first pharmaceutical companies in the 19th century started as fine chemicals businesses. The discovery of penicillin introduced antibiotics, and improved synthetic techniques led to the giants we know as big pharma today. Today, in the 21st century both computing and biotechnology has allowed for great leaps forward in precision medicine. Biotechnology refers to the manipulation of living organisms or their components to produce useful commercial products. In the pharmaceutical industry this refers to genetic engineering for novel pharmaceuticals.
Here, we focus on the use of recombinant technology to create proteins for use in biomedical applications. Recombinant proteins are proteins formed by laboratory methods of molecular cloning. Through this technology, we are able to elucidate sequence-structure-function relationships of proteins, and determine their specific functions. Additionally, recombinant methods allow us to fine tune or modify the sequences of natural proteins to be more effective scaffolds or reagents.
Chapter 3 focuses on the development of synthetic proteins for medical diagnostics. We designed a protein scaffold, based on natural innate immunity proteins, to detect bacteria cell wall components. Chapter 4 focuses on the engineering of keratin protein with applications in wound healing. We introduce controlled degradation of the biomaterial for use in potential drug delivery systems at the wound site. Chapter 5 focuses on the use of recombinant technologies aiding in the elucidation of a regulatory protein's function in cell division.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/107278 |
| Date | 06 July 2020 |
| Creators | Kim, Christina Sue Kyung |
| Contributors | Chemistry, Etzkorn, Felicia A., Santos, Webster L., Esker, Alan R., Schulz, Michael |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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