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master's thesis
Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97
Zagreb: University of Zagreb, Faculty of Science, 2023. urn:nbn:hr:217:838249
Bezjak, Tea
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

Cite this document

Bezjak, T. (2023). Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97 (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:838249

Bezjak, Tea. "Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:838249

Bezjak, Tea. "Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:838249

Bezjak, T. (2023). 'Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97', Master's thesis, University of Zagreb, Faculty of Science, accessed 28 March 2025, https://urn.nsk.hr/urn:nbn:hr:217:838249

Bezjak T. Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97 [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2023 [cited 2025 March 28] Available at: https://urn.nsk.hr/urn:nbn:hr:217:838249

T. Bezjak, "Pročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:217:838249

Metadata
TitlePročišćavanje i kristalizacija proteinskog kompleksa SPRTN:p97
Title (english)Purification and crystallization of the protein complex SPRTN:p97
AuthorTea Bezjak
MentorNives Ivić (mentor)
Committee memberMarko Močibob (predsjednik povjerenstva)
Committee memberIvica Đilović (član povjerenstva)
Committee memberAdriana Kenđel (član povjerenstva)
Committee memberŽeljka Car (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2023-02-23, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
DNA-ovisna metaloproteaza Spartan (SPRTN) održava genomski integritet sudjelujući u popravku križnih veza DNA i proteina (engl. DNA-protein crosslink, DPC), čestog tipa oštećenja DNA. Citotoksičnost DPC-ova pripisuje se ometanju staničnih procesa koji uključuju DNA (replikacija, transkripcija i popravak) te predstavljaju prepreku u održavanju genomske stabilnosti. Pokazano je da SPRTN sudjeluje u vezanju AAA+ ATP-aze p97, čija je najpoznatija uloga odmotavanje ubikvitinitanih... More proteina. Rješavanje kristalne strukture kompleksa SPRTN:p97 i njegova strukturna karakterizacija omogućila bi dublje razumijevanje uloge p97 u popravku DPC oštećenja. Stoga su humani SPRTN i p97 te njihovi skraćeni konstrukti, prekomjerno eksprimirani i pročišćeni kromatografskim metodama. Metodom povlačenja proteina okarakterizirana je interakcija navedenog kompleksa. Kako bi se pokušalo odrediti stehiometriju nastalog kompleksa napravljena je elektroforeza na poliakrilamidnom gelu u nativnim uvjetima. Kompleks proteina SPRTN i p97 kristaliziran je metodom difuzije para u svrhu rješavanja njihove kristalne strukture. Inicijalni kristalizacijski uvjeti su detaljno optimizirani kako bi se pripremili monokristali zadovoljavajuće veličine i kvalitete, što bi u konačnici omogučilo rješavanje kristalne strukture kompleksa SPRTN:p97. Less
Abstract (english)
The DNA-dependent metalloprotease Spartan (SPRTN) functions in maintaining genomic integrity by participating in the repair of DNA-protein crosslinks (DPCs), a common type of DNA damage. The cytotoxicity of DPCs is attributed to the disruption of cellular processes involving DNA (e.g. replication, transcription and repair) and poses a challenge in the maintenance of genomic stability. It was shown that SPRTN participates in the binding of the AAA+ ATPase p97, well known for... More its function in unwinding of ubiquitinated proteins. Solving the crystal structure of the SPRTN:p97 complex and its functional characterization would allow a deeper understanding of the p97 role in DPC damage repair. Therefore, human SPRTN and p97, as well as several of their deletion mutants were overexpressed and purified by standard chromatography techniques. Subsequently, complex formation between SPRTN and p97 was tested and characterized via pull-down assays. To estimate the stoichiometry of the complex, native polyacrylamide gel electrophoresis was applied. SPRTN and p97 complex was subjected to crystallization by vapor diffusion method. Initial crystallization conditions were further optimized with the aim to obtain larger and higher quality diffracting crystals. Taken together, this set the stage for obtaining the structure of the SPRTN:p97 complex. Less
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:838249
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Embargo expiration date: 2025-02-23
Terms of useLicence In copyright icon
Created on2023-03-01 12:05:02
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