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Title: The Incorporation of Azide-bearing Unnatural Amino Acids into Proteins for Bio-orthogonal Reactions
Authors: Abdeljabbar, Diya Maher
Advisors: Link, Aaron J
Contributors: Chemical and Biological Engineering Department
Subjects: Chemical engineering
Issue Date: 2012
Publisher: Princeton, NJ : Princeton University
Abstract: Unnatural amino acid (UAA) incorporation has become the method of choice for many protein engineering endeavors. UAAs can be introduced into recombinant proteins via residue-specific or site-specific incorporation methods. While site-specific incorporation is useful for making point mutations that result in minimal changes in the overall structure, residue-specific incorporation permits engineering of the overall physical and chemical properties of proteins by globally replacing a natural amino acid. The work described in this thesis harnesses the power of residue-specific incorporation to endow proteins with reactive chemical handles that can be modified by azide-alkyne cycloaddition (click chemistry). Since aminoacyl-tRNA synthetases (aaRS) determine which amino acid gets incorporated into a cellular protein, these enzymes can be engineered to enable the integration of various UAAs into recombinant proteins. A gene that encodes for a particular engineered aaRS is commonly placed on a plasmid in addition to the native aaRS gene found in the host organism's genome. In Chapters 2 and 3, the gene encoding for an engineered methionyl-tRNA synthetase (metG*) was integrated into the genome of a methionine-auxotrophic and prototrophic strain of E. coli in place of the native metG allele. The protein product of this gene, referred to as MetRS-L13G, was previously engineered to incorporate the reactive UAA, azidonorleucine (ANL). Even with only a single genomic copy of metG*, methionine-auxotrophic E. coli was able to integrate ANL into recombinant proteins with a 90% extent of incorporation. Remarkably, the engineered methionine-prototrophic E. coli was able to incorporate ANL at 42%, which is 2.5-fold higher than the wild-type strain with a plasmid-borne copy of metG*. The work performed in Chapters 2 and 3 highlights the fact that the single genomic copy of metG* can fully support cell growth under methionine-rich conditions, yet also endow proteins with an azide-functionality that is useful for bioconjugation via azide-alkyne cycloaddition. In addition to using azide-bearing amino acids and click chemistry for bioconjugation applications, Chapter 4 of this thesis describes the use of UAA incorporation in order to constrain protein secondary structures. The UAAs azidohomoalanine (AHA) and para-ethynylphenylalanine (PEP) were both incorporated into recombinant proteins in E. coli. By engineering these UAAs into proteins, the azide and alkyne side chains of the novel amino acids were capable of crosslinking via click chemistry. This crosslink between the two UAAs is shown to stabilize the alpha-helical structure of a leucine zipper protein at elevated temperatures, as shown by circular dichroism. In addition, a larger protein, barstar, was also incorporated with both UAAs in order to create a crosslink at the interior region of the protein. This experiment provides evidence that this method of "protein stapling" can be extended to even larger proteins. While Chapters 2-4 take advantage of the fact that UAAs can be incorporated into bacteria, Chapter 5 demonstrates that residue-specific incorporation can be extended to a multicellular organism. This chapter highlights how AHA can be incorporated into the nematode, C. elegans, without any genetic manipulation of the host. Moreover, the data shows that the endogenous C. elegans proteins, which have been endowed with bioorthogonal chemical handles, can be used as click chemistry substrates with the potential for various applications. The body of work described in this thesis underscores the utility of UAA incorporation when combined with click chemistry. As a result of the wealth of research being done in this area, a valuable toolbox has been developed for the incorporation of a diverse set of UAAs that can be used in a wide variety of application-based research.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemical and Biological Engineering

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