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master's thesis
Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza
Zagreb: University of Zagreb, Faculty of Science, 2024. urn:nbn:hr:217:646127
Knežević, Karla
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

Cite this document

Knežević, K. (2024). Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:646127

Knežević, Karla. "Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza." Master's thesis, University of Zagreb, Faculty of Science, 2024. https://urn.nsk.hr/urn:nbn:hr:217:646127

Knežević, Karla. "Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza." Master's thesis, University of Zagreb, Faculty of Science, 2024. https://urn.nsk.hr/urn:nbn:hr:217:646127

Knežević, K. (2024). 'Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza', Master's thesis, University of Zagreb, Faculty of Science, accessed 30 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:646127

Knežević K. Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2024 [cited 2024 December 30] Available at: https://urn.nsk.hr/urn:nbn:hr:217:646127

K. Knežević, "Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2024. Available at: https://urn.nsk.hr/urn:nbn:hr:217:646127

Metadata
TitleOptimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza
Title (english)Optimization of expression and purification of atypical arginyl-tRNA-synthetases
AuthorKarla Knežević
MentorIta Gruić Sovulj (mentor)
Committee memberIta Gruić Sovulj (predsjednik povjerenstva)
Committee memberIvana Kekez (član povjerenstva)
Committee memberIva Juranović Cindrić (član povjerenstva)
Committee memberMorana Dulić (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2024-09-05, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Arginil-tRNA-sintetaza (ArgRS) je enzim koji katalizira nastajanje arginil-tRNA, supstrata za biosintezu proteina, u dva koraka: aktivacija arginina pomoću ATP-a te njegovo prenošenje na tRNAArg. Razred aminoacil-tRNA-sintetaza (aaRS) kojemu pripada ArgRS karakterizira očuvana struktura katalitičke domene s dva kanonska motiva, His-hidrofobna aminokiselina-Gly-His (HXGH) i Lys-Met-Ser-Lys-Ser (KMSKS), koji sudjeluju u interakciji s ATP-om te adenilatnim međuproduktom. U literaturi su opisane aaRS s atipičnim motivom HXGH koje posjeduju poboljšana svojstva poput hiperrezistencije na antibiotik. U sklopu ovog diplomskog rada odabrana su dva organizma čije su se atipične ArgRS proučavale: Symbiobacterium thermophilum čija ArgRS umjesto kanonskog motiva HXGH posjeduje motiv Val-Leu-Val-Gln (VLVQ), te Herpetosiphon aurantiacus u čijem se genomu nalaze dva gena za ArgRS: jedan posjeduje kanonski motiv His-Val-Gly-His (HVGH) dok drugi protein ima atipični motiv Arg-Val-Asp-His (RVDH). Svrha ovog istraživanja je optimizacija ekspresije i pročišćavanja arginil-tRNA-sintetaza s atipičnim motivom HXGH te proučavanje kinetike enzimski katalizirane reakcije aminoacilacije. Pronađeni su uvjeti optimalne ekspresije i pročišćavanja ArgRS iz S. thermophilum i H. aurantiacus (HVGH). Proteini aminoaciliraju pripadnu tRNAArg sličnom brzinom. Atipična ArgRS iz H. aurantiacus (RVDH) sklona je agregaciji te nije uspješno pročišćena i kinetički okarakterizirana.
Abstract (english)
Arginyl-tRNA synthetase (ArgRS) catalyzes the formation of arginyl-tRNA, a substrate for protein biosynthesis by a two-step mechanism: ATP-dependant activation of arginine which is followed by arginine transfer to tRNA. The class of aminoacyl tRNA synthetases (aaRS) to which ArgRS belongs is characterized by a conserved catalytic fold containing two canonical motifs which interact with ATP and the adenylate intermediate, His-hydrophobic aminoacid- Gly-His (HXGH) and Lys-Met-Ser-Lys-Ser (KMSKS). Research shows that the aaRS containing the atypical HXGH motif possesses enhanced properties such as hyper-resistance to antibiotics. This research aims to find the optimal conditions for the expression and purification of atypical ArgRS from Symbiobacterium thermophilum and Herpetosiphon aurantiacus. The former's ArgRS possesses the Val-Leu-Val-Gln (VLVQ) motif and the latter's genome has two genes for ArgRS. The primary ArgRS has the canonical His-Val-Gly-His (HVGH) motif but the secondary has the atypical Arg-Val-Asp-His (RVDH) motif. Optimal conditions for expression and purification of S. thermophilum and canonical ArgRS from H. aurantiacus were found. Kinetic characterisation shows that these enzymes have similar aminoacylation rates. H. aurantiacus ArgRS with the atypical RVDH motif is prone to foming soluble aggregates and hence was not successfully purified and kinetically characterized.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:646127
ProjectNumber: IP-2022-10-1400
Title: Istraživanja aminoacil-tRNA-sintetaza kao antibiotskih meta i mehanizama kojima razvijaju rezistenciju
Title: Investigation of aminoacyl-tRNA syntetases as targets for antibiotics and how they develop resistance mechanisms
Acronym: ARSTARE
Leader: Ita Gruić-Sovulj
Jurisdiction: Croatia
Funder: Hrvatska zaklada za znanost
Funding stream: Research Projects
Study programmeTitle: Graduate university studies in chemistry; specializations in: Graduate university studies in chemistry
Course: Graduate university studies in chemistry
Study programme type: university
Study level: graduate
Academic / professional title: sveučilišni/a magistar/magistra kemije (sveučilišni/a magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2024-10-10 13:56:46