Priprava in selekcija knjižnice afiteles z namenom prekinitve homodimerizacije proteaze MPro

Oven, Eva (avtor), Novinec, Marko (mentor)

Glavna proteaza virusa SARS-CoV-2 (MPro) ima ključno vlogo pri replikaciji virusa, saj je nujna za procesiranje virusnih poliproteinov. Njena encimska aktivnost je odvisna od tvorbe dimera, kar odpira možnost alternativnega terapevtskega pristopa, ki temelji na zaviranju dimerizacije namesto neposrednega ciljanja katalitičnega mesta encima. Cilj magistrskega dela je bil pripraviti knjižnico različnih variant afiteles in med njimi najti afitelo, sposobno prekinitve homodimerizacije proteaze MPro. Uspešnost prekinitve smo detektirali v bakterijskem sistemu Escherichia coli za detekcijo homodimerizacije proteinov. V ta namen je bil obstoječi sistem LexGFP nadgrajen v sistem LexKan, pri katerem je prekinitev homodimerizacije povezana z izražanjem odpornosti na kanamicin. Sistem omogoča selekcijo bakterij, v katerih je prišlo do prekinitve dimerizacije proteaze MPro s pripravljenimi afitelesi. S kloniranjem po Gibsonu smo pripravili ustrezne plazmidne konstrukte in uspešno vzpostavili izboljšan sistem za detekcijo homodimerizacije LexKan. Z dvema zaporednima kloniranjema po Gibsonu smo pripravili plazmid pAFI, ki vsebuje zaporedje afitelesa in selekcijski marker za odpornost proti kloramfenikolu. Plazmid je služil kot matrica za pripravo knjižnice afiteles, ki smo jo pripravili z uporabo degeneriranih podaljšanih oligonukleotidov. Afitelesa so tako vsebovala naključne mutacije na značilnih aminokislinskih mestih vezavne površine. Selekcijo knjižnice smo izvedli s sistemom LexKan. Negativna kontrola v sistemu LexKan je potrdila, da je mogoče zaznati homodimerizacijo proteinskih tarč v bakterijskem okolju. Čeprav v okviru te naloge nismo identificirali afitelesa, ki bi učinkovito prekinilo dimerizacijo MPro, smo pokazali, da je priprava knjižnice afiteles z degeneriranimi podaljšanimi oligonukleotidi izvedljiva. Hkrati smo identificirali več možnih izboljšav, ki bi jih bilo treba uvesti za uspešno selekcijo. Predpostavljamo da ima sistem LexKan, ki temelji na himerah transkripcijskih faktorjev LexA, potencialno širšo uporabnost pri preučevanju proteinskih interakcij ter pri razvoju novih terapevtskih strategij, usmerjenih v zaviranje dimerizacije tarčnih proteinov.

• Jezik: Slovenski jezik
• Ključne besede: afitelesa, MPro, homodimerizacija, LexA sistem, proteinske interakcije
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-188484
• Datum objave v RUL: 23.09.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Preparation and selection of an affibody library to disrupt the homodimerization of the MPro protease

Izvleček

The main protease of SARS-CoV-2 (MPro) plays a key role in viral replication, as it is essential for the processing of viral polyproteins. Its enzymatic activity depends on the formation of a homodimer, which provides an opportunity for alternative therapeutic strategies based on inhibiting dimerization rather than directly targeting the catalytic site of the enzyme. The aim of this master’s thesis was to prepare a library of different affibody variants and to identify an affibody capable of disrupting the homodimerization of the MPro protease. The ability to interfere with dimerization was assessed using a bacterial Escherichia coli system designed for the detection of protein homodimerization. For this purpose, the previously established LexGFP system was modified to generate the LexKan system, in which disruption of homodimerization results in the expression of kanamycin resistance. This setup allows for the selection of bacterial cells in which MPro dimerization has been disrupted by the tested affibodies. Appropriate plasmid constructs were prepared using Gibson cloning, and an improved LexKan system for monitoring homodimerization was successfully established. The pAFI plasmid, containing the affibody sequence and a chloramphenicol resistance marker, was constructed through two consecutive Gibson cloning steps. This plasmid was used as a template for the generation of an affibody library using degenerate megaprimers, introducing random mutations at selected amino acid positions on the binding surface. Library selection was subsequently performed using the LexKan system. The results showed that the LexKan system is suitable for detecting homodimerization of protein targets in a bacterial environment, as demonstrated by the appropriate negative control. Although no affibody capable of efficiently disrupting MPro dimerization was identified within the scope of this work, the study demonstrates that the generation of an affibody library using degenerate megaprimers is feasible. In addition, several aspects of the system were identified that could be optimized to improve future selection efficiency. The LexKan system, which is based on chimeric LexA transcription factors, therefore represents a promising tool for studying protein–protein interactions and may be applicable to the development of therapeutic strategies targeting protein dimerization.

• Ključne besede: affibodies, MPro, homodimerization, LexA system, protein-protein interactions