رسالة جامعية
Cloning, expression, purification and enzyme kinetics characterization of deubiquitinase USP30
| العنوان: | Cloning, expression, purification and enzyme kinetics characterization of deubiquitinase USP30 |
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| المؤلفون: | Flores, Rosemary |
| بيانات النشر: | University of Delaware |
| سنة النشر: | 2016 |
| المجموعة: | The University of Delaware Library Institutional Repository |
| مصطلحات موضوعية: | Cysteine proteinases, Cysteine proteinases -- Purification, Enzymes, Catalysis, Serine, Chemical kinetics, Escherichia coli, Molecular cloning, Gene expression |
| الوصف: | Deubiquitinating enzymes (DUBs) are imperative participants in multiple cellular processes, and as a consequence the control of DUB function has become the subject of great interest due to its possible implications in neurodegenerative diseases, anti-viral/bacterial responses, and potential anti-cancer targets. Recently a new DUB, USP30, was shown to be localized in the mitochondria and identified as having a role in the regulation of mitochondrial mitophagy, whose dysfunction is associated with the neurodegenerative Parkinson’s disease. The ubiquitin specific proteases (USPs), cysteine proteases, have a conserved catalytic core including cysteine, histidine and aspartate/asparagine. USP30 is very similar to other DUBs, except for the Asp/Asn conserved residue which is a serine. Our efforts were to understand how this unconventional catalytic residue in the catalytic core plays a role in the control of deubiquitination by first characterizing its steady state kinetics. A truncated USP30 was cloned into a pSUMO vector for expression in E.coli to obtain the wild-type (WT) turnover rate. Mutations in the catalytic triad were made to further understand the influence of the serine in the catalytic core. The WT kcat/KM was estimated to be 3.1 x 104 M-1s-1, while the C77A USP30 mutant had no detectable activity, as expected, and the S477A USP30 mutation had an estimated kcat/ KM of 2.2 x102 M-1s -1. Our results give insight to the importance of the serine and its possible role in regulating the rate of the USP30. ; Zhuang, Zhihao ; M.S. ; University of Delaware, Department of Chemistry and Biochemistry |
| نوع الوثيقة: | thesis |
| وصف الملف: | application/pdf |
| اللغة: | unknown |
| ردمك: | 978-1-86850-830-3 1-86850-830-7 |
| العلاقة: | https://search.proquest.com/docview/1868508307?accountid=10457Test; 993259643; http://udspace.udel.edu/handle/19716/21508Test |
| الإتاحة: | http://udspace.udel.edu/handle/19716/21508Test |
| رقم الانضمام: | edsbas.11E74966 |
| قاعدة البيانات: | BASE |
| ردمك: | 9781868508303 1868508307 |
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