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Brian_Marshall_Masters_Thesis.pdf (1.41 MB)

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Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein

Marshall, Brian A
http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988

Abstract Details

2018, Master of Science, Ohio State University, Chemical Engineering.
Current chromatographic techniques are unable to purify recombinant proteins in an easy, reliable, cost-effective manner without modifying the amino acid sequence and using proteolytic enzymes to remove these modifications, making the process not scalable due to the expense of these enzymes. Inteins are a promising alternative to this approach, as they have been engineered over the past two decades to perform a cleaving reaction capable of producing tag-less recombinant proteins. A significant issue is that the rate at which this reaction occurs at different rates depending on the protein that is being identified. To address this issue, this study performed saturation mutagenesis on the first amino acid immediately after the end of the ¿I-CM intein to characterize the kinetics of the intein and determine what properties of the amino acid residues exhibited a significant effect on the cleaving rate. The study showed that certain amino acids cleaved so quickly that there was over 50% pre-cleavage for those residues (e.g. serine, histidine, methionine, and cysteine) and others cleaved very slowly, with over 50% precursor after 24 hours (valine and proline). The amino acid properties that were determined to be significant molecular weight, volume, hydrophobicity, isoelectric point, charge, pKa, hydropathy index, hydrogen bonding characteristics, and if the residue as aliphatic, aromatic, or sulfur-containing.
David Wood, Ph.D. (Advisor)
Jeffery Chalmers, Ph.D. (Committee Member)
44 p.
Chemical Engineering

Recommended Citations

Citations

  • Marshall, B. A. (2018). Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988

    APA Style (7th edition)

  • Marshall, Brian. Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein. 2018. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988.

    MLA Style (8th edition)

  • Marshall, Brian. "Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein." Master's thesis, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988

    Chicago Manual of Style (17th edition)

osu1532363837193988
191
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This open access ETD is published by The Ohio State University and OhioLINK.