Razvoj sistema za zaznavanje heterodimerizacije proteina na osnovi ločene molekule GFP

Makuc, Nika (author), Novinec, Marko (mentor)

Zeleni fluorescenčni protein (GFP) je protein, ki zaradi prisotnosti kromofora ob osvetlitvi z modro svetlobo oddaja fluorescenco zelene barve. Molekulo GFP lahko razdelimo na dva ločena fragmenta, ki ju lahko v obliki fuzije pripnemo na poljubna proteina. V primeru, da označena proteina med seboj interagirata, se fragmenta GFP spontano združita v funkcionalen protein, sposoben fluorescence. Na ta način je mogoče zaznavati in kvantificirati specifične interakcije med proteini z merjenjem intenzitete fluorescence. Cilj naloge je bil razviti sistem za zaznavanje heterodimerizacije proteinov na osnovi ločene molekule GFP v bakterijskih celicah. Sistem smo zasnovali z vnosom kasete, ki vsebuje zapis za tarčna proteina v obliki fuzije z ustreznima fragmentoma GFP, v plazmid biRNA. Za tarčna proteina smo izbrali človeška proteina p53 in MDM2, ki medsebojno interagirata in tvorita heterodimer. Na N- in C-konec zapisa za oba tarčna proteina smo vnesli kombinaciji kompatibilnih restrikcijskih mest, ki omogočajo enostavno zamenjavo tarčnih proteinov. Detekcija heterodimerizacije v razvitem sistemu omogoča hkratno merjenje intenzitete fluorescence in optične gostote bakterijskih kultur. Delovanje sistema za zaznavanje heterodimerizacije smo preverili na modelnem primeru interakcije med človeškima proteinoma p53 in MDM2 ter potrdili z uporabo pozitivne in negativne kontrole. Kot pozitivno kontrolo smo uporabili C-končno domeno proteina LexA, ki tvori homodimer, kot negativno kontrolo pa protein MBP, ki je monomer. Rezultati so potrdili pravilno delovanje sistema, saj je bila pri izražanju proteinov p53 in MDM2 ter pri proteinu LexA zaznana povečana intenziteta fluorescence, kar je posledica heterodimerizacije oz. homodimerizacije in ponovne združitve fragmentov GFP. Nasprotno pa pri proteinu MBP, ki ne dimerizira, povečanja intenzitete fluorescence nismo zaznali. Vitalnost in sposobnost delitve celic smo dodatno potrdili z merjenjem optične gostote, s čimer smo izključili možnost, da bi bila povečana intenziteta fluorescence posledica povečane gostote celic.

• Language: Slovenian
• Keywords: Heterodimerizacija, GFP, ločena molekula GFP, p53, MDM2
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186413
• Publication date in RUL: 01.09.2026

Secondary language

• Language: English
• Title: Development of a system for detecting protein heterodimerization based on split GFP

Abstract

Green fluorescent protein (GFP) is a protein that emits green fluorescence upon excitation with blue light due to the presence of a chromophore. The GFP molecule can be split into two separate fragments, which can be fused to proteins of interest. When the tagged proteins interact, the GFP fragments spontaneously reassemble into a functional fluorescent protein. This approach enables the detection and quantification of specific protein–protein interactions by measuring fluorescence intensity. The aim of this study was to develop a system for detecting protein heterodimerization based on split GFP in bacterial cells. We designed the system by introducing a cassette containing the coding sequences for the target proteins as fusions with the respective GFP fragments into the biRNA plasmid. As the target proteins we used human proteins p53 and MDM2, which interact with each other to form a heterodimer. At the N- and C-termini of the coding sequences for both target proteins, we incorporated combinations of compatible restriction sites to allow easy replacement of the target proteins. Detection of heterodimerization in the developed system enables simultaneous measurement of fluorescence intensity and optical density of bacterial cultures. The functionality of the system was evaluated using the interaction between the human proteins p53 and MDM2 as a model system and comfirmed using positive and negative controls. As a positive control, we used the C-terminal domain of the LexA protein, which forms a homodimer, and as a negative control, we used the MBP protein, which is monomeric. The results confirmed proper system function, as an increase in fluorescence intensity was observed for p53 and MDM2 and LexA, reflecting heterodimerization and homodimerization, respectively, and the subsequent reassembly of GFP fragments. In contrast, no increase in fluorescence was detected for MBP, which does not dimerize. Cell viability and proliferation were further confirmed by optical density measurements, ruling out increased cell density as a cause of elevated fluorescence intensity.

• Keywords: Heterodimerisation, GFP, split GFP, p53, MDM2