Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification

Stimple, Samuel Douglas
http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Chemical Engineering.
The development of platform technologies can dramatically impact the speed and ease with which new biological processes/products can be optimized and brought to market. Platform technologies offer researchers predictable, generalizable procedures for achieving a pre-defined outcome. This dissertation describes the development of two platforms for i) engineering RNA-based tools for gene regulation and ii) engineering self-cleaving intein affinity purification tags with more tightly-controlled cleavage kinetics for recombinant protein purification. Bacterial trans- acting small regulatory RNAs (sRNAs) have great potential for applications in the field of metabolic engineering, due to their modular nature and the relative ease with which they may be engineered for novel regulatory function at the level of mRNA translation control. Furthermore, these sRNAs act at their target mRNAs through relatively simple base-pairing interactions, and (in many cases) have been demonstrated to be portable from microbe to microbe, while also providing the benefit of tuning gene expression of multiple genes for optimization of metabolic pathway flux. Chapter 2 describes the development of a genetic system for engineering novel, multi-acting sRNA regulators derived from the E. coli-native DsrA sRNA. Chapter 3 establishes thorough design rules for these engineered sRNAs to ensure robust regulation of the target gene, and describes a design basis that provides these semi-synthetic sRNAs with unprecedented specificity for their target mRNAs. Chapter 4 describes the validation and use of a previously-established, yeast-surface display-based protein evolution platform for the engineering of self-cleaving intein affinity tags with applications in bioseparations. Affinity tag technology greatly simplifies the process of purifying diverse recombinantly-expressed proteins, but tag removal remains a non-trivial barrier to implementation of affinity capture for protein purification at scale. Affinity tags can be genetically fused to self-cleaving inteins to simplify the tag removal process. Following protein capture via the tag, autocatalytic cleavage of the intein releases a tagless target protein. However, premature cleavage of the intein during protein expression severely limits the utility of this technology by rendering intein-mediated purification platforms incompatible with expression in mammalian hosts and by diminishing overall process recoveries. To address these shortcomings, a directed evolution approach was combined with a yeast-surface display-based screen for generating inteins with highly-controllable cleavage kinetics. Inteins with greatly improved control over their cleavage kinetics were identified, and should serve as a useful basis for further evolutionary optimization of this useful technology.
David Wood (Advisor)
Jeffrey Chalmers (Committee Member)
Andre Palmer (Committee Member)
199 p.
Chemical Engineering
protein purification, intein, protein engineering, self-cleaving affinity tag, sRNAs, metabolic engineering, gene regulation, noncoding sRNA, synthetic biology

Recommended Citations

Citations

  • Stimple, S. D. (2018). Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145

    APA Style (7th edition)

  • Stimple, Samuel. Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145.

    MLA Style (8th edition)

  • Stimple, Samuel. "Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145

    Chicago Manual of Style (17th edition)

osu1514494485801145
128
© 2018, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.