magistrsko delo
Neža Pavko (Avtor), Helena Gradišar (Mentor), Miha Pavšič (Član komisije za zagovor), Marko Dolinar (Član komisije za zagovor), Gregor Gunčar (Komentor)

Povzetek

Proteinski origami iz ovitih vijačnic je eden izmed pristopov de novo načrtovanja proteinov, ki temelji na modularnosti in daljnosežnih interakcijah med posameznimi segmenti proteina. Do sedaj so bili uspešno načrtovani in okarakterizirani proteinski origamiji v velikosti do 100 kDa v obliki tetraedra, štiristrane piramide, bipiramide in tristrane prizme. Pri načrtovanju še večjih proteinskih origamijev v velikosti nekaj 100 kDa je glavna omejitev omejen nabor seta ortogonalnih ovitih vijačnic, ki predstavljajo osnovo nanokletk. Cilj tega magistrskega dela je bil razviti novo strategijo načrtovanja večjih struktur na osnovi do sedaj razvitih struktur proteinskega origamija in možnosti predstavitev proteinskih domen na njihovih ogliščih. Proteinski origami v obliki tetraedra TET12SN smo nadgradili z integracijo sistema SpyCatcher/SpyTag, ki ima sposobnost tvorbe izopeptidne vezi. Sprva smo načrtovali osnovne gradnike sistema, pri čemer smo oglišča TET12SN dekorirali z eno, dvema, tremi ali štirimi domenami SpyCatcher ali SpyTag. Vsi konstrukti, dekorirani s proteinsko domeno SpyCatcher v ogliščih, so ohranili katalizno aktivnost, ki jo ima prosta domena SpyCatcher, medtem ko je konstrukti z domenami SpyTag, vezanih na istih mestih, niso. Zato smo eksperimentalno identificirali optimalna mesta za vstavitev proteinske domene SpyTag z ozirom na ohranjanje aktivnosti in tako načrtovali TET12SN, dekoriran z eno, dvema, tremi ali štirimi domenami SpyTag. Po uspešni izolaciji in karakterizaciji vseh osnovnih konstruktov z analitsko kromatografijo z ločevanjem po velikosti, meritvami cirkularnega dikroizma, sipanjem rentgenskih žarkov pri nizkih kotih in nativno poliakrilamidno gelsko elektroforezo smo nadaljevali s sestavljanjem kompleksnih struktur. Pri tem smo med seboj kombinirali različne osnovne konstrukte, kjer smo za centalno ogrodje uporabili osnovni konstrukt z različnim številom domen SpyCatcher ali SpyTag, kot ligandno ogrodje pa osnovni konstrukt z eno parno domeno - SpyTag ali SpyCatcher. Osnovni gradniki so se povezali z izopeptidnimi vezmi, nastale komplekse pa smo okarakterizirali z analitsko kromatografijo z ločevanjem po velikosti, nativno poliakrilamidno gelsko elektroforezo, poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata ter sipanjem rentgenskih žarkov pri nizkih kotih. Uspešno smo potrdili nastanek in obliko mono-, di-, tri- in tetrakonjugiranih kompleksov, pri čemer je bilo osrednje ogrodje dekorirano s proteinsko domeno SpyCatcher ali SpyTag. Tako načrtovani kompleksi so največji do sedaj opisani proteinski origamiji, pri čemer je molekulska masa najmanjšega tovrstnega okarakteriziranega konjugiranega kompleksa 170 kDa, masa največjega pa presega 400 kDa. Sistem, razvit tekom magistrskega dela, bi v nadaljevanju lahko nadgradili z integracijo drugih komponent (npr. nanoteles, antigenov, encimov ali drugih vezavnih domen) in na ta način naredili korak bližje funkcionalizaciji proteinskega origamija za medicinske ali industrijske aplikacije.

Ključne besede

ovite vijačnice;proteinski origami;proteinske domene;SpyCatcher;SpyTag;magistrska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [N. Pavko]
UDK: 577.112(043.2)
COBISS: 176568323 Povezava se bo odprla v novem oknu
Št. ogledov: 67
Št. prenosov: 7
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: 'Formation of complex structures from coiled-coil protein origami tetrahedron nanocages with SpyCatcher/SpyTag system
Sekundarni povzetek: Coiled-coil protein origami (CCPO) is one of the de novo approaches to protein design, which is based on modularity and long-range interactions between the individual segments of a protein. So far, protein origami smaller than 100 kDa in the form of tetrahedra, tetrahedral pyramids, bipyramids and trihedral prisms have been successfully designed and characterised. When designing even larger protein origami of a few 100 kDa, the main hurdle is the limited size of our set of orthogonal coiled-coils that are the foundation of the CCPO. The aim of this MSc thesis was to develop a new strategy for the design of larger structures based on already developed structures of CCPO and the ability of protein domain presentation at their vertices. We upgraded the protein origami in the form of the TET12SN tetrahedron by integrating the SpyCatcher/SpyTag system, which has the ability to form isopeptide bonds. Initially, we designed the basic building blocks of the system by decorating the vertices of TET12SN with one, two, three or four SpyCatcher or SpyTag domains. All constructs decorated with the SpyCatcher protein domain retained the same catalytic activity as the free SpyCatcher domain, whereas SpyTag domains at the same insertion sites did not retain activity. Therefore we experimentally identified optimal sites for the insertion of the SpyTag protein domain with respect to activity retention, and thus designed TET12SN decorated with one, two, three or four SpyTag domains. After successful isolation and characterisation of all the basic constructs by analytical size exclusion chromatography, circular dichroism spectroscopy, small-angle X-ray scattering and native polyacrylamide gel electrophoresis, we proceeded to the formation of complex structures. Here, we combined different basic constructs, using a basic construct with a different number of SpyCatcher or SpyTag domains as the central scaffold and a basic construct with a single copy of a suitable pair, SpyTag or SpyCatcher, as the ligand scaffold. The basic building blocks were linked via isopeptide bonds and the resulting complexes were characterised by analytical size exclusion chromatography, native polyacrylamide gel electrophoresis, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and small-angle X-ray scattering by which we successfully confirmed the formation and shape of mono-, di-, tri- and tetraconjugated complexes. The complexes designed in this way are the largest protein origami described to date, with the smallest such characterised conjugated complex having a molecular mass of 170 kDa and the largest one exceeding 400 kDa. The system developed in the course of the MSc thesis could be further upgraded by the integration of other components (e.g. nanobodies, antigens, enzymes or other binding domains), thus taking a step closer to the functionalisation of protein origami for medical or industrial applications.
Sekundarne ključne besede: protein origami;coiled-coils;SpyCatcher;SpyTag;Beljakovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 1000377
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani: 108 str.
ID: 20799415