Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein
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The Ohio State University / OhioLINK
2018
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu15323638371939882021-08-03T07:07:53Z Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein Marshall, Brian A. 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. 2018 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988 http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Chemical Engineering |
| spellingShingle |
Chemical Engineering Marshall, Brian A. Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| author |
Marshall, Brian A. |
| author_facet |
Marshall, Brian A. |
| author_sort |
Marshall, Brian A. |
| title |
Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| title_short |
Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| title_full |
Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| title_fullStr |
Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| title_full_unstemmed |
Immediate C-terminal Extein Effects on the Cleaving Kinetics of the ¿I-CM Mini-Intein |
| title_sort |
immediate c-terminal extein effects on the cleaving kinetics of the ¿i-cm mini-intein |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2018 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1532363837193988 |
| work_keys_str_mv |
AT marshallbriana immediatecterminalexteineffectsonthecleavingkineticsoftheicmminiintein |
| _version_ |
1719454504201486336 |