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Title: Development of Designer Proteins Using Split Inteins
Authors: Mostafavi, Anahita
Advisors: Muir, Tom W
Contributors: Molecular Biology Department
Keywords: Amber suppression
Expanded genetic code
Split inteins
Subjects: Biochemistry
Issue Date: 2019
Publisher: Princeton, NJ : Princeton University
Abstract: Protein splicing is a process in which an intervening protein (intein) excises itself from a host protein (extein), while simultaneously ligating the two ends in which it is embedded. The study of protein splicing has led to the development of a number of protein engineering technologies. In this work, we developed split-intein based methods to generate designer proteins with (1) multiple non-canonical amino acids (ncAAs) and (2) de novo repeat sequences to manufacture bio-polymers. Most proteins undergo post-translational modification (PTM) following ribosomal synthesis, and these PTMs have important effects on protein folding, stability, localization and catalytic activity. The ability to incorporate ncAAs into proteins has enabled researchers to examine diverse structural and electronic variants at specific locations, modifying the peptide backbone, and probing the effects of PTMs, leading to a number of biological discoveries. Current methods used to incorporate multiple site-specific ncAAs into a single protein are limited by size, number of possible incorporation sites, low efficiency, and/or laborious methodology. By combining amber suppression with protein trans-splicing, we developed an approach to produce proteins with multiple modifications. In this method, the protein of interest (POI) is split into two fragments such that the modification sites are separated, and these fragments are fused to the halves of a split intein. The two transcripts, with amber codons, are expressed, purified, and spliced to produce the full-length dually-modified protein. This approach allows the production of homo- and/or heterotypic dually-modified proteins with greater efficiency than current accessible methods. In addition to developing improved methods for producing modified proteins, we employed split inteins to generate proteins with repetitive sequence elements. Materials made up of repeating units of short peptides are critical in many biological processes such as signaling, structure, and molecular recognition. While these polymers have the potential to be repurposed for a variety of applications, they are notoriously difficult to produce using standard protein expression methods. To overcome these difficulties, we used a single split intein to generate de novo protein polymers in vivo and orthogonal inteins to produce protein fibers larger than 250 kDa in vitro. These methods allow for the production of previously impossible designer proteins.
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:Molecular Biology

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