Tvorba dimernih proteinskih nanokletk z uporabo sistema SpyCatcher/SpyTag in proteinskega origamija

Pavko, Neža (author), Jerala, Roman (mentor)

Naravni proteini so biopolimeri različnih dolžin sestavljeni iz kombinacij 20 osnovnih aminokislin. Vseh možnih kombinacij aminokislinskih zaporedij, ki določajo različne oblike in funkcije proteinov, je bistveno več, kot jih najdemo v naravi. To je povod za oblikovanje drugačnih, v naravi še neodkritih proteinskih struktur z de novo sintezo proteinov, katere predstavnik je tudi proteinski origami z obvitimi vijačnicami. Proteinski origami lahko služi kot ogrodje za predstavljanje različnih proteinskih domen in posledično tvorbo kompleksnejših proteinskih struktur. V ta namen smo uporabili sistem SpyCatcher/SpyTag, za katerega je značilna spontana tvorba izopeptidne vezi. Cilj diplomske naloge je bil sinteza konstrukta, ki ima v enem od oglišč predstavljeno proteinsko domeno SpyCatcher, kot rekombinanten protein pripravljen z aerobnim bioprocesom v E.coli. Temu je sledila izolacija proteina z NiNTA afinitetno kromatografijo in gelsko filtracijo ter analiza le-tega z NaDS-PAGE. Pripravljen protein smo nato kombinirali s proteinoma SpyTag-RFP in Tet12SN-StStStR, ki vsebujeta eno oziroma tri domene SpyTag. Z analizo Native-PAGE in SEC-MALS smo uspešno potrdili aktivnost proteina Tet12SN-ScGG, ki je tvoril monokonjugat oz. dimer s SpyTag-RFP. Pri reakciji Tet12SN-ScGG s Tet12SN-StStStR pa je prišlo do nastanka mono-, di- in tri-konjugatov. Z eksperimentalnim delom smo potrdili hipotezo, da je s pomočjo domen SpyCatcher in SpyTag, predstavljenih v ogliščih proteinskih tetraedrov, mogoče tvoriti dimerno oziroma mono-konjugirano strukturo, še več, uspešno smo pripravili strukture sestavljene iz treh in štirih tetraedrskih podenot.

• Language: Slovenian
• Keywords: bionanotehnologija, proteinski origami, obvite vijačnice, SpyCatcher, SpyTag
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Pavko]
• Year: 2020
• PID: 20.500.12556/RUL-117309
• UDC: 604.2:661.745:579.254.2(043.2)
• COBISS.SI-ID: 21894147
• Publication date in RUL: 05.07.2020

Secondary language

• Language: English
• Title: Formation of dimeric protein nanocages with the SpyCatcher/SpyTag system and protein origami

Abstract

Natural proteins are biopolymers of different lengths which are constructed form 20 amino acids. There are many possible amino acid sequence combinations which determine proteins' shapes and functions, yet only a limited amount of those can be found in nature. Constructing new and unseen proteins with different functions is the main goal of de novo protein design. One of many approaches to de novo protein design is coiled coil protein origami (CCPO). Protein origami can be used as a frame for attachment of different protein domains which can lead to obtaining more complex protein structures. For this purpose we used the SpyCatcher/SpyTag system that is known for its spontaneous isopeptide bond formation ability. The main goal of this thesis is the synthesis of a tetrahedral protein which has attached one SpyCatcher domain, named Tet12SN-ScGG. The protein was isolated with NiNTA affinity chromatography and size exclusion chromatography followed by NaDS-PAGE analysis. Isolated protein was then combined with proteins SpyTag RFP and Tet12SN-StStStR, which have one and three SpyTag domains attached, respectively. With Native-PAGE and SEC-MALS analysis we successfully confirmed activity of Tet12SN-ScGG, which formed monoconjugates/dimers with SpyTag- RFP. Reactions between Tet12SN-ScGG and Tet12SN-StStStR led to the formation of mono- di- and tri-conjugates. We confirmed our hypothesis that formation of dimeric protein nanocages with the use of the SpyCatcher/SpyTag system is possible – furthermore, we successfully constructed a structure, composed of four tetrahedral subunits.

• Keywords: bionanotechnology, protein origami, coiled-coils, SpyCatcher, SpyTag