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Title: Discovery, Design, and Deployment of Enhanced Split Inteins
Authors: Stevens, Adam Joseph
Advisors: Muir, Tom W
Contributors: Chemistry Department
Keywords: intein splicing
protein design
protein engineering
protein semisynthesis
Subjects: Chemistry
Issue Date: 2018
Publisher: Princeton, NJ : Princeton University
Abstract: Protein splicing is a post-translational autoprocessing event in which an intervening protein (intein) spontaneously cleaves itself from a precursor protein while simultaneously ligating the adjacent residues (exteins) to form a native peptide bond. Due to the efficiency of this ligation reaction, inteins have found widespread use as tools in protein engineering and chemical biology. Naturally split inteins, which are separately expressed and then undergo association and splicing in trans, are of particular interest. However, applications of split inteins have been limited by a number of shortcomings, including issues of expression yield among protein-intein fusions and decreased splicing rates under certain extein contexts (extein dependency). Through structural and mechanistic-guided approaches, we have engineered split inteins with improved robustness and extein promiscuity. We utilized consensus design to engineer a consensus fast (Cfa) split DnaE intein that exhibits unprecedented thermal and chaotropic stability and a consensus atypical (Cat) split intein that is the fastest atypical split intein reported to date. In addition, targeted saturation mutagenesis was applied to engineer extein promiscuity into DnaE inteins. Lastly, to address the tendency of split intein fusion proteins to aggregate, a general strategy using intrasteric stabilization was employed in which an appended peptide sequence acts as an internal chaperone for split intein and extein fragments. These enhanced split inteins were then applied to a number of protein engineering methods, such as the generation of head to tail cyclized proteins, the modification of a monoclonal antibody with a small molecule cargo, and the semisynthesis of cellular chromatin in isolated nuclei. Furthermore, the increased stability of Cat compared to previously reported atypical split inteins enabled the first mechanistic investigation of atypical split intein association. Overall, we expect these enhanced inteins to improve current intein-based technologies and encourage the further use and development of protein trans- splicing as a tool for protein engineering.
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:Chemistry

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