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Hybrid-Phase Native Chemical Ligation Approaches to Overcome the Limitations of Protein Total Synthesis

Yu, Ruixuan Ryan

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Total protein synthesis allows the preparation of proteins with chemically diverse modifications. The numerous advantages of total synthesis are sometimes offset by some major limitations. Protein synthesis is a non-trivial task involving many chemical steps, and these steps increase with the size of the protein. Therefore, larger proteins are difficult to synthesize with high yield. We have developed a strategy which we term hybrid-phase native chemical ligation (NCL) to overcome some of the limitations of size and yield. Hybrid-phase NCL combines ligating peptides on a solid support (solid-phase NCL) and in solution (solution-phase NCL) to maximize synthetic yield. We have successfully used this method to synthesize triple-acetylated histone H4-K5ac,K12ac,K91ac and, for the first time, acetylated centromeric histone CENP-A-K124ac (CpA-K124ac). In order to improve the yield of CENP-A total synthesis, we have incorporated a convergent ligation element in our hybrid-phase strategy. This new approach reduced the number of purification steps, leading to a synthetic yield that was almost triple that of the original approach. Finally, we introduce the convergent solid-phase hybrid NCL approach that allows the preparation of a long peptide segment bearing a masked thioester on a solid support. Through a newly developed resin-anchoring strategy, cleavage of the product from solid-phase generated a ligation-compatible segment that could be used directly with no purification. This method has the potential to synthesize large proteins in good yield, effectively overcoming the size and yield limits of protein total synthesis.
Jennifer Ottesen (Advisor)
Dennis Bong (Committee Member)
Michael Freitas (Committee Member)
Michael Poirier (Committee Member)
214 p.

Recommended Citations

Citations

  • Yu, R. R. (2016). Hybrid-Phase Native Chemical Ligation Approaches to Overcome the Limitations of Protein Total Synthesis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469191712

    APA Style (7th edition)

  • Yu, Ruixuan. Hybrid-Phase Native Chemical Ligation Approaches to Overcome the Limitations of Protein Total Synthesis. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469191712.

    MLA Style (8th edition)

  • Yu, Ruixuan. "Hybrid-Phase Native Chemical Ligation Approaches to Overcome the Limitations of Protein Total Synthesis." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469191712

    Chicago Manual of Style (17th edition)