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Developing Molecular Tools for Applications in Metabolic Engineering and Protein Purification

lahiry, ashwin
http://rave.ohiolink.edu/etdc/view?acc_num=osu1502109789024711

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Microbiology.
Biotechnology is an important field of science that utilizes the principles of biology and engineering to build applications in multi-disciplinary research areas. This dissertation discusses the development of molecular tools in two such research areas: 1) industrial biotechnology and 2) medical biotechnology. Chapters 2 and 3 of this dissertation focus on the development of a multi-targeting small regulatory RNA (sRNA) as a potentially useful tool for metabolic engineering applications in industrial biotechnology. sRNAs can govern gene expression by base-pairing with the translation initiation regions (TIRs) of mRNAs. These sRNAs can be engineered to target the TIRs of non-cognate mRNAs by altering their antisense sequences. Due to their modular, tunable and portable nature, they possess unique regulatory qualities for optimizing metabolic pathways. In Chapter 2, an Escherichia coli-based genetic system was created for the development of dual-acting sRNA variants. These variants were built on the DsrA scaffold, a well-characterized multi-acting E. coli sRNA and assayed for the regulation of two separate TIRs fused to independent reporter genes. Expression of the sRNA and each reporter mRNA was independently controlled, and regulatory effects were quantified in vivo using a microtiter plate assay. Using this system, semi-rationally designed DsrA variants were screened in E. coli for their ability to simultaneously regulate two key TIRs from the Clostridium acetobutylicum n-butanol synthesis pathway. In Chapter 3, this system was utilized to successfully design and test the functionality of DsrA variants in two industrially relevant microorganisms, Bacillus subtilis and Clostridium acetobutylicum. This study demonstrates the value of the genetic system and the sRNA scaffold for designing DsrA variants that specifically target desired mRNAs, and thus provides a platform for retargeting and characterizing multi-acting sRNAs for metabolic engineering applications. Chapter 4 discusses the development of a self-cleaving affinity tag for the purification of recombinant proteins and has applications in medical biotechnology. Process development of new recombinant biotherapeutic proteins involves complex optimization and scale up based on the characteristics of each protein. There is no simple, low-cost platform that can be utilized to purify these diverse proteins. Thus, it is desirable to create an affinity tag-based platform for purification of any recombinant protein, but with the requirement that the purified protein be tagless and traceless. Consequently, our lab has developed a pH-controllable self-cleaving tag based on the Npu DnaE intein from Nostoc punctiforme for affinity-based purification of recombinant proteins. Previous work done with self-cleaving inteins has shown that the target protein residues (extein residues) at the cleavage junction can strongly affect cleavage kinetics of the tag. Therefore, in Chapter 4, the extein dependence of this tag is characterized using model (eGFP) and biotherapeutic (streptokinase (SK) and granulocyte colony stimulating factor (GCSF)) proteins. Through these studies, N-terminal extein residues that result in accelerated or diminished cleavage kinetics were identified. An eGFP model system was also established to predict the effect of the primary extein sequence on the cleavage kinetics of this tag. Finally, the information from this study was utilized to improve cleavage kinetics of the tag with GCSF.
David Wood, Dr. (Advisor)
217 p.
Microbiology
Biotechnology; sRNA engineering; metabolic engineering; self-cleaving tags; protein purification platform; inteins

Recommended Citations

Citations

  • lahiry, A. (2017). Developing Molecular Tools for Applications in Metabolic Engineering and Protein Purification [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1502109789024711

    APA Style (7th edition)

  • lahiry, ashwin. Developing Molecular Tools for Applications in Metabolic Engineering and Protein Purification. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1502109789024711.

    MLA Style (8th edition)

  • lahiry, ashwin. "Developing Molecular Tools for Applications in Metabolic Engineering and Protein Purification." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1502109789024711

    Chicago Manual of Style (17th edition)

osu1502109789024711
313
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This open access ETD is published by The Ohio State University and OhioLINK.