Predstavitev proteinskih domen fluorescenčnih proteinov in luciferaze na ogrodju proteinskega origamija iz obvitih vijačnic v obliki tetraedra.

Tušar, Anja (author), Jerala, Roman (mentor)

Tetraedrične kletke, ki jih tvorijo obvite vijačnice, so uspešen primer načrtovanja proteinskih nanomaterialov. Cilj magistrske naloge je bil načrtovati, pripraviti in izolirati tetraedrične proteine, ki imajo na vsa oglišča vezano Renilla luciferazo in zeleni fluorescenčni protein v različnih kombinacijah. Za povečanje fleksibilnosti strukture in s tem produkcije topnega proteina smo luciferazne domene vezali na oglišča preko tri, šest ali devet aminokislinskega povezovalnega zaporedja. Sintetizirane proteine smo očistili z Ni-NTA kromatografijo in gelsko izključitveno kromatografijo. Iz SEC kromatogramov smo razbrali, da je v vzorcih konstruktov s tri-aminokislinskim povezovalnim zaporedjem nižja koncentracija monomernega proteina, kot pri enakih konstruktih s šest aminokislin dolgim povezovalcem. Konstrukti z devet-aminokislinskim povezovalcem so bili izolirani v prenizki koncentraciji za nadaljno karakterizacijo. Uspešno izoliranim proteinskim konstruktom smo določili velikost, delež monomernega proteina in vsebnost sekundarnih struktur z NaDS-PAGE elektroforezo, SEC-MALS in cirkularnim dikroizmom. Konstrukti so imeli najvišjo topnost, najvišji delež monomernega proteina in so bili izolirani v najvišji koncentraciji, če je bila luciferaza na ogrodje vezana preko šest aminokislin dolgega povezovalnega zaporedja. Izolirani proteinski konstrukti so bili pričakovane velikosti in so imeli CD spektre značilne za strukture, ki so večinsko sestavljene iz alfa vijačnic. Dokazali smo uspešno izolacijo tetraedričnega ogrodja z vezanimi domenami luciferaze in zelenega fluorescenčnega proteina.

• Language: Slovenian
• Keywords: bionanotehnologija, proteinski origami, obvite vijačnice, zeleni fluorescenčni protein, Renilla luciferaza
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Tušar]
• Year: 2019
• PID: 20.500.12556/RUL-111069
• UDC: 601.4:577.21(043.2)
• COBISS.SI-ID: 9350265
• Publication date in RUL: 22.09.2019

Secondary language

• Language: English
• Title: Design of coiled coil tetrahedral protein origami fused with fluorescence and luciferase protein domains

Abstract

Tetrahedral shaped cages built from concatenated coiled-coils are a successful example of protein nanotechnology. Master thesis project aimed to design and isolate tetrahedral proteins with Renilla luciferase and fluorescent protein domains fused on all of its vertices. Three, six or nine amino acids long linkers were added to luciferase domain to adjust the flexibility of introduced folded domain and improve production of soluble protein. Purification of produced proteins was done with Ni-NTA and size-exclusion chromatography. SEC results showed that protein constructs with three amino acids linkers have lower fraction of monomeric protein in the sample than the same constructs with six amino acid linkers. The amount of purified protein construct with nine amino acid linkers was insufficient for protein characterization. Protein size, fraction of the monomeric form and the secondary structures content were determined by SDS-PAGE electrophoresis, SEC-MALS and circular dichroism. Constructs with six amino acid linkers were most soluble with the highest fraction of monomeric protein and were isolated at the highest concentration among the produced constructs. The isolated proteins had expected sizes and CD spectra demonstrated high content of alfa helices. We have demonstrated that it is possible to successfully isolate coiled-coil tetrahedral origami fused with fluorescence and luciferase protein domains in their vertices.

• Keywords: bionanotechnology, protein origami, coiled-coil, green fluorescent protein, Renilla luciferase