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Moody Thesis Final.pdf (1.38 MB)

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Impact of a mutation known to improve Npu intein splicing activity on an engineered cleaving variant of the intein

Moody, Nathan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1629152122786765

Abstract Details

2021, Master of Science, Ohio State University, Chemical Engineering.
Bridging the gap between protein purification platforms at the laboratory scale and at the industrial scale remains a challenge, as there is no “one size fits all” technology that works for every protein. To date, only technology based on Protein A has managed to enter mainstream usage both in the lab and in industry, but it only works for monoclonal antibody and Fc fusions. Affinity chromatography can be used to purify most proteins, but a major downside of this is that they require the use of affinity tags – proteins purified with these tags must undergo further treatment to remove the tags, which is impractical at the industrial scale. Research into intein-based chromatography offers a possibility to get around this: engineered inteins can be used as a “self-cleaving” affinity tag that leaves a tagless target protein after elution. However, the rate at which inteins cleave themselves off a protein varies wildly depending on the target protein. This work aims to reduce that dependency by introducing a mutation known to reduce the intein’s dependence on the +2 C-extein residue in the natural splicing version of the Npu intein into the engineered cleaving version to determine if it can also reduce its dependency on the +2 residue. The experiments performed in this work did not detect a significant difference in variance, however. In addition, it also appeared to slow down the overall cleavage kinetics, suggesting that the intein cleavage reaction is subject to different controls than the splicing reaction.
Andre Palmer (Committee Member)
David Wood (Advisor)
67 p.
Chemical Engineering
split intein; intein cleavage; protein engineering; protein purification; chromatography

Recommended Citations

Citations

  • Moody, N. (2021). Impact of a mutation known to improve Npu intein splicing activity on an engineered cleaving variant of the intein [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1629152122786765

    APA Style (7th edition)

  • Moody, Nathan. Impact of a mutation known to improve Npu intein splicing activity on an engineered cleaving variant of the intein. 2021. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1629152122786765.

    MLA Style (8th edition)

  • Moody, Nathan. "Impact of a mutation known to improve Npu intein splicing activity on an engineered cleaving variant of the intein." Master's thesis, Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1629152122786765

    Chicago Manual of Style (17th edition)

osu1629152122786765
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This open access ETD is published by The Ohio State University and OhioLINK.
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