magistrsko delo
Martina Lokar (Author), Gregor Gunčar (Mentor), Marko Dolinar (Thesis defence commission member), Miha Pavšič (Thesis defence commission member)

Abstract

Sintetični proteini, ki jih lahko pridobimo s preoblikovanjem obstoječih nativnih proteinov ali z de novo sintezo, se v zadnjih letih zaradi svojih lastnosti vedno pogosteje uveljavljajo v številnih biotehnoloških in terapevtskih aplikacijah. Umetno oblikovani proteini s specifično tridimenzionalno strukturo in funkcijo so med drugim pomembni kot modulatorji imunskega sistema, samosestavljajoči se biomateriali ali kot senzorji v raznolikih biotehnoloških sistemih. Njihovo načrtovanje in sinteza omogočata tudi boljše razumevanje osnovnih principov proteinskega zvijanja in povezav med tridimenzionalno strukturo ter funkcijo proteina. Primer sintetičnega proteina je Pizza α vijačnici, ki je zgrajen iz tretjine proteina Pizza2-SR z β propelerskim zvitjem in svežnja dveh α-vijačnic proteina DF1-L13A. Na podlagi predvidene strukture proteina Pizza-α-vijačnici predpostavljamo, da proteinski monomeri med seboj spontano interagirajo in tvorijo samosestavljajoč se heksamerni kompleks v obliki tristrane prizme, imenovan tudi proteinska kletka Pizza α vijačnici. Do interakcij med monomeri prihaja s trimerizacijo regije z β-propelerskim zvitjem, sočasno pa v prisotnosti dvovalentnih kovinskih ionov antiparalelno dimerizirata tudi svežnja dveh α-vijačnic. Cilj magistrskega dela je bil pripraviti plazmidni konstrukt z ustreznim zapisom za sintetičen protein Pizza α vijačnici, v nadaljevanju protein izraziti v bakterijah in ga iz bakterijskih celic izolirati v čisti obliki. Z verižno reakcijo s polimerazo (PCR) smo uspeli pripraviti dva konstrukta proteina Pizza-α-vijačnici (Pizza 2hx-AM in Pizza 2hx bM), ki smo ju v nadaljevanju izrazili in izolirali s kovinskokelatno afinitetno kromatografijo (IMAC). V drugem delu smo na osnovi kromatografije z ločevanjem po velikosti (SEC) dokazali, da je protein Pizza 2hx AM pri različnih pufrskih pogojih v ravnotežju med več različnimi oligomernimi stanji. Določili smo, da je največ heksamernega kompleksa v raztopini prisotnega ob dodatku nizke koncentracije kelatorja kovinskih ionov EDTA. Te ugotovitve smo dodatno potrdili z nativno poliakrilamidno gelsko elektroforezo (nativni PAGE), prečnim povezovanjem z glutaraldehidom in sledečo poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata (NaDS PAGE) ter z določanjem molekulske mase proteinskih kompleksov na podlagi multidetektorskega sistema. S SEC smo tudi določili, da proteinski konstrukt Pizza 2hx-bM ne oblikuje heksamernega kompleksa. Na koncu smo želeli predvideno tridimenzionalno strukturo heksamera Pizza α-vijačnici dokončno potrditi z rentgensko kristalografijo, vendar nam pri kristalizacijskem eksperimentu ni uspelo pridobiti dovolj velikih kristalov, da bi bili primerni za rentgensko kristalografsko analizo.

Keywords

proteinske kletke;sintetični protein Pizza-▫$\alpha$▫-vijačnici;bionanomateriali;metaloproteini;sveženj vijačnic;oligomerni kompleksi;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Lokar]
UDC: 577.112(043.2)
COBISS: 129923331 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Expression and characterization of synthetic self-assembly protein cage composed of α-helix bundles and β-propeler fold
Secondary abstract: Synthetic proteins can be created by remodelling existing native proteins or by a de novo protein synthesis. Because of their characteristics, these proteins are frequently used in many biotechnological and therapeutical applications. Designer proteins with specific three-dimensional structure and function may be useful as modulators of the immune system, as self-assembling biomaterials or as sensors in different biotechnological systems. Furthermore, the process of artificial protein modelling and synthesis allows us to better understand the basic principles of protein folding and also the relationship between three-dimensional protein structure and protein function. Pizza α helices is a synthetic protein composed of one-third of Pizza2-SR protein with β propeller fold and a two-helix bundle from DF1-L13A protein. Based on the predicted structure of Pizza α helices, we anticipate a spontaneous intermolecular interaction between Pizza α helices protein monomers, which consequently leads to the formation of a self assembling hexamer complex in the shape of a triangular prism named Pizza α helices protein cage. Individual monomers interact through trimerisation of protein region with β-propeller fold and also through antiparallel dimerisation of two two helix bundles in the presence of divalent metal ions. The purpose of my work was to prepare a plasmid construct with a correct nucleotide sequence of Pizza α helices synthetic protein, to express the designed protein in expression bacterial strain and finally to isolate the protein from bacterial cells. Using polymerase chain reaction (PCR), we prepared two different Pizza α helices constructs (Pizza-2hx-AM and Pizza-2hx-bM), which were extracted from bacterial cultures by immobilised metal affinity chromatography (IMAC). After that, using size-exclusion chromatography (SEC), we proved the presence of an equilibrium between different Pizza-2hx-AM oligomeric complexes in distinct buffer solutions. We could determine that the highest quantity of Pizza α helices hexamer was detected in a buffer solution containing a low concentration of a divalent metal ion chelator EDTA. These findings were additionally confirmed with native polyacrylamide gel electrophoresis (Native PAGE), glutaraldehyde crosslinking followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by determination of the molecular weight of protein complexes using multi-detector system. Using SEC, we have also determined that protein construct Pizza-2hx-bM does not form a hexamer complex. Finally, we wanted to confirm the predicted three dimensional structure of the Pizza α helices hexamer complex using X-ray crystallography. Unfortunately, in the protein crystallisation experiment, we were not able to grow protein crystals, which would be adept for X-ray analysis.
Secondary keywords: synthetic protein Pizza-▫$\alpha$▫-helices;protein cage;oligomeric complex;Beljakovine;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 79 str.
ID: 16411130