diplomsko delo
Ela Bizjak (Author), Gregor Gunčar (Mentor)

Abstract

Cisteinska proteazna domena (CPD) izhaja iz večfunkcijskega samoprocesirajočega toksina, ki vsebuje ponavljajoče motive (MATRX), iz bakterije Vibrio cholerae. Domena je odgovorna za virulentnost toksina, saj s cepitvijo na več mestih aktivira ostale domene, med njimi tudi domeno, ki kovalentno povezuje aktin in Rho-inaktivirajočo domeno. Za aktivacijo mora priti CPD v stik z inozitol heksakisfosfatom (IP6), ki je prisoten v citosolu evkariontskih celic. Ker CPD specifično cepi tudi na mestu, ki je del njenega aminokislinskega zaporedja, je primerna za uporabo kot samoizrezujoči se označevalec. Prednosti njene uporabe so: eliminacija potrebe po uporabi komercialno dostopnih proteaz, izvedba cepitve na koloni za nikljevo afinitetno kromatografijo in nizki stroški uporabe IP6. V diplomskem delu smo v bakteriji Escherichia coli seva BL21[DE3] izražali protein EGFP s C-končno fuzijo CPD s heksahistidinsko oznako. Z opravljenim poskusnim izražanjem smo določili, da zvijanje proteina ob indukciji z IPTG najbolje poteka pri 18 ⁰C. Izražanje smo ob takih pogojih izvajali tudi v večjem merilu. Rekombinantni protein smo preko heksahistidinske oznake izolirali, ga v prisotnosti različnih koncentracij IP6 aktivirali in opazovali, kako uspešno poteka samoizrezovanje. Cepitev smo spremljali v raztopini in preko vezave na aktivirane nikljeve kroglice. Potrdili smo, da prezgodnja aktivacija in nespecifične cepitve niso prisotne. Optimalne rezultate smo dosegli ob dvourni inkubaciji na aktiviranih nikljevih kroglicah s 500 μM IP6 pri 18 ⁰C. Za nadaljnjo karakterizacijo proteina smo po kromatografiji z ločevanjem po velikosti izvedli kristalizacijske preglede in dosegli kristalizacijo neaktivirane CPD v obliki proteina EGFP-CPD-His6.

Keywords

samoizrezujoči se označevalci;cisteinska proteazna domena;CPD;MARTX;inozitol heksakisfosfat;ojačani zeleni fluorescenčni protein;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [E. Bizjak]
UDC: 577.112:577.21(043.2)
COBISS: 132882691 Link will open in a new window
Views: 59
Downloads: 16
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Other data

Secondary language: English
Secondary title: Efficiency of self-cleaving CPD tag from bacteria Vibrio cholerae
Secondary abstract: Cysteine protease domain (CPD) derives from multifunctional autoprocessing repeats-in-toxin (MARTX) of Vibrio cholerae. The domain is responsible for the virulence of the toxin, as it cleaves on multiple points, thus activating other domains, such as the actin crosslinking and Rho-inactivating domains. For its activation, CPD needs to be induced by inositol hexakisphosphate (IP6), present in the eukaryotic cell cytosol. As CPD specifically cleaves at the site in its own amino acid sequence, it is possible to use it as a self-cleaving protein tag. Its advantages are: eliminating the need to use commercial proteases, the ability to induce the cleavage on histidine trap and the low cost of using IP6. As part of our research, we used bacteria Escherichia coli strain BL21[DE3] to express an EGFP protein with C-end CPD fusion with a hexahistidine tag. Through test expression, we determined that protein folding, when induced with IPTG, is best at 18 ⁰C. We applied the same conditions for large-scale expression. We isolated the recombinant protein by hexahistidine tag, activated it with different concentrations of IP6 and monitored if self-cleavage was successful. We observed the cleavage in free solution and while bonded to activated nickel-agarose beads. We confirmed that premature activation and unspecific cleavage is not present. The best results were achieved with two-hour incubation on activated nickel-agarose beads with 500 μM IP6 at 18 ⁰C. To further characterize the protein, after purifying it with size exclusion chromatography, we performed several crystallization screens and crystallized inactive CPD in the form of EGFP-CPD-His6 protein.
Secondary keywords: self-cleaving tag;cysteine protease domain;CPD;MARTX;inositol hexakisphosphate;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 35 str.
ID: 16706506