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Merideth Cooper Dissertation.pdf (7.85 MB)

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Creating an Efficient Biopharmaceutical Factory: Protein Expression and Purification Using a Self-Cleaving Split Intein

Cooper, Merideth A
http://orcid.org/0000-0001-5397-4923
http://rave.ohiolink.edu/etdc/view?acc_num=osu152226172238882

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Recombinant therapeutic proteins changed the world over 30 years ago when insulin, the first therapeutic protein, was approved. Since then, over 200 therapeutic proteins have been approved to treat a wide range of diseases from diabetes to immune disorders. Currently, there is no universal platform that can be used to purify any given target protein in a quick and inexpensive manner. The self-cleaving split intein tag technology remedies this issue by creating a universal platform that can purify any traceless, tagless target protein rapidly and economically. Chapter 2 discusses the combination of the split intein purification strategy with cell-free protein synthesis (CFPS) systems to reduce the time it takes to produce therapeutic proteins. With the cell-free systems, proteins can be produced in hours compared to days or even weeks. The combination of CFPS and the split intein tag technology has been utilized in the creation of a device to produce biologics on demand. The BioMOD device aims to produce a single-dose of any therapeutic protein within 24 hours, specifically with a military application in mind. Chapter 3 discusses the use of magnetic beads to mediate the split intein purification. Combining the split intein and magnetic beads creates a more efficient purification process that requires less buffer and set-up time. Four target proteins are used to demonstrate the applicability of the system. Chapter 4 discusses the regeneration of a commercially available resin that has been used to covalently immobilize the N fragment of the split intein using a thioester bond. Due to the commercially available resin having a high price point and the lengthy amount of time it takes to immobilize the N fragment, regeneration of the resin was necessary. A panel of buffers was screened to find the best regeneration buffer. Using the best buffer, a life cycle analysis was done using 20 regeneration cycles to show the resin could be regenerated multiple times. The development of a split intein resin that can be regenerated and reused multiple times makes this platform technology more realistic. Chapter 5 summarizes all the work discussed and goes into future work that can be done on each of the projects including further optimization of CFPS and the creation a split intein that is alkaline stable for regeneration using sodium hydroxide.
David Wood (Advisor)
Jeffrey Chalmers (Committee Member)
Andre Palmer (Committee Member)
175 p.
Chemical Engineering
Protein purification, intein, Nostoc punctiforme DnaE intein, protein engineering, split intein, magnetic beads, intein purification, cell-free protein synthesis, IVT

Recommended Citations

Citations

  • Cooper, M. A. (2018). Creating an Efficient Biopharmaceutical Factory: Protein Expression and Purification Using a Self-Cleaving Split Intein [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu152226172238882

    APA Style (7th edition)

  • Cooper, Merideth. Creating an Efficient Biopharmaceutical Factory: Protein Expression and Purification Using a Self-Cleaving Split Intein . 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu152226172238882.

    MLA Style (8th edition)

  • Cooper, Merideth. "Creating an Efficient Biopharmaceutical Factory: Protein Expression and Purification Using a Self-Cleaving Split Intein ." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu152226172238882

    Chicago Manual of Style (17th edition)

osu152226172238882
624
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This open access ETD is published by The Ohio State University and OhioLINK.