Izražanje in izolacija skrajšanih oblik človeškega proteina FHL2

Šegota, Luka (author), Gaber, Aljaž (mentor)

FHL2 (angl. Four and a half LIM domain protein 2) je adapterski protein, ki je sestavljen le iz štirih in pol domen LIM. Ta je poimenovana po treh genih, pri katerih je bila domena prvič opisana (LIN-11, Isl-1 in MEC-3). Sestavljena je iz dveh zaporednih cinkovih prstov, ki vsak koordinirata vezavo enega Zn2+ iona. Domene LIM nimajo katalitske aktivnosti, vendar se povezujejo z mnogimi proteini, kot so strukturni proteini, receptorji, signalni proteini in transkripcijski partnerji. Zaradi tako širokega nabora vezavnih partnerjev ni čudno, da so proteini, ki vsebujejo domeno LIM, povezani z mnogimi bolezenski stanji. FHL2 sodeluje v mnogih signalnih poteh, med drugim je koaktivator poti Wnt, ki regulira številne celične procese. Glavni mediator te signalne poti je protein β-katenin, ki v tem primeru interagira s FHL2, a je mehanizem te interakcije slabo poznan. V okviru diplomskega dela smo želeli bolje spoznati vlogo posameznih domen FHL2 pri interakcijah s proteini, zato smo načrtali knjižnico 14-ih skrajšanih oblik, ki vsebujejo le eno (LIM0, LIM1, LIM2, LIM3, LIM4), dve (LIM0-1, LIM1-2, LIM2-3, LIM3-4), tri (LIM0-2, LIM1-3, LIM2-4) ali štiri domene (LIM0-3, LIM1-4). Zapise za proteine smo sestavili z metodo PCR, na N-konec pa smo jim dodali heksahistidinsko oznako (His6) in zeleni fluorescenčni protein (sfGFP), ki ju je mogoče odcepiti s proteazo TEV. Zapise smo vključili v tarčni vektor pET-32b(+) in jih izrazili v bakterijskih celicah E. coli. V nadaljevanju smo izrazili in očistili pet proteinov, ki vsebujejo le po eno domeno proteina, pri čemer je bil postopek izražanja in čiščenja enak že prej optimiziranemu protokolu pridobivanja celotnega FHL2. Morebitno interakcijo med posameznimi domenami in β-kateninom smo analizirali z gelsko izključitveno kromatografijo, sklopljeno z detektorjem za analizo statičnega sipanja laserske svetlobe pri majhnem in pravem kotu (LALS/RALS). Tako zastavljena knjižnica konstruktov bo omogočala vpogled v vlogo domen pri interakciji z ostalimi vezavnimi partnerji, kar bo prispevalo k razumevanju mehanizma delovanja FHL2 v bioloških procesih. Postopek čiščenja celotnega proteina se je izkazal za optimalnega tudi pri pridobivanju posameznih domen proteina, a kljub temu so se domene na določene korake čiščenja odzvale različno. Z analizo vezave smo potrdili, da nobena izmed petih domen FHL2 ni dovolj za stabilno reakcijo z β-kateninom, vendar bi za boljši vpogled morali preveriti še interakcijo z ostalimi proteini, ki vsebujejo več domen.

• Language: Slovenian
• Keywords: FHL2, β-katenin, proteinska interakcija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-139822
• COBISS.SI-ID: 128037123
• Publication date in RUL: 07.09.2022

Secondary language

• Language: English
• Title: Expression and isolation of truncated forms of human FHL2 protein

Abstract

FHL2 (Four and a half LIM domain protein 2) is an adapter protein that consist of only four and a half LIM domains. The domain is named after the three genes, where it was initially described (LIN-11, Isl-1 and MEC-3). It consists of two consecutive zinc fingers, each coordinating the binding of one Zn2+ ion. LIM domains do not have catalytic activity, but they associate with many proteins, such as structural proteins, receptors, signalling proteins and transcription partners. Due to such a wide range of binding partners, it is not surprising that proteins containing the LIM domain are associated with many disease states. FHL2 participates in many signalling pathways, among others it is a coactivator of the Wnt pathway, which regulates many cellular processes. The main mediator of this signalling pathway is a protein called β-catenin, which in this case interacts with FHL2, but the binding mechanism is poorly understood. As part of the thesis, we wanted to better understand the role of individual FHL2 domains in interactions with proteins, so we constructed a library of 14 truncated forms, which contain one (LIM0, LIM1, LIM2, LIM3, LIM4), two (LIM0-1, LIM1-2, LIM2-3, LIM3-4), three (LIM0-2, LIM1-3, LIM2-4) or four domains (LIM0-3, LIM1-4). Transcripts for the proteins were assembled by the PCR technique, with hexa histidine-tag (His6) and green fluorescent protein (sfGFP) added to the N-terminus of transcripts, but both can be cleaved by TEV protease. The transcripts were inserted into the targeting vector pET-32b(+) and expressed in E. coli cells. Next, we expressed and purified five proteins containing only one FHL2 domain each, furthermore the expression and purification procedure was the same as the previously optimized protocol for obtaining the whole FHL2. The possible interaction between the individual domains and β-catenin was analysed by size-exclusion chromatography coupled with a detector for the analysis of static laser light scattering at low and right angles (LALS/RALS). The assembled library of constructs will provide insight into the role of the domains in the interaction with other binding partners, which will contribute to the understanding of FHL2 in biological processes. The purification process of the entire protein proved to be optimal in obtaining individual domains of the protein, but even so, the domains responded differently to certain purification steps. Through the binding analysis, we confirmed that none of the five domains of FHL2 is sufficient for a stable reaction with β-catenin, but for better insight the interaction with other proteins that contain more domains should be analysed.

• Keywords: FHL2, β-catenin, protein interaction