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Control of Intein-Mediated Self-Cleaving Tag for Recombinant Protein Purification

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Recent advances in upstream bioprocessing have largely increased the productivities of recombinant protein expression in various expression systems. This has led to an increasingly urgent need for rapid, cost-effective and large-scale downstream unit operations. Affinity tag-based purification provides an efficient platform to purify virtually any uncharacterized protein in a highly selective, high-throughput manner. With the combination of a self-cleaving intein, the purification and the tag removal processes can all be carried out in a single chromatography step, thus dramatically reducing the cost of downstream processing. Over the past decades, inteins have been genetically modified for better controllability and cleaving ability. However, unwanted in vivo premature intein cleaving remains a major bottleneck for applications in mammalian systems. In this dissertation, recent progresses in the control of intein cleaving will be discussed. A continuous delta I-CM intein and a zinc binding motif fusion allow the intein-tagged precursor proteins to be expressed in mammalian systems with less than 40-50% in vivo premature cleaving in the presence of 200 micro molar zinc chloride. An alternative, the Npu split intein system, has been shown to completely eliminate premature cleaving and has been altered into a pH sensitive cleaving intein. These efforts to control intein cleaving have led to the development of a robust and efficient platform technology for non-antibody recombinant protein downstream purification.
David Wood (Advisor)
Andre Palmer (Committee Member)
Shang-Tian Yang (Committee Member)
Werner Tajrks (Committee Member)
179 p.

Recommended Citations

Citations

  • Han, T.-C. (2016). Control of Intein-Mediated Self-Cleaving Tag for Recombinant Protein Purification [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1453206997

    APA Style (7th edition)

  • Han, Tzu-Chiang. Control of Intein-Mediated Self-Cleaving Tag for Recombinant Protein Purification. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1453206997.

    MLA Style (8th edition)

  • Han, Tzu-Chiang. "Control of Intein-Mediated Self-Cleaving Tag for Recombinant Protein Purification." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1453206997

    Chicago Manual of Style (17th edition)