Izražanje in izolacija transpeptidaznih encimov, vpletenih v biosintezo peptidoglikana

Mesarič, Katja (avtor), Hrast Rambaher, Martina (mentor), Cingl, Jernej (komentor)

Bakterijska odpornost proti protibakterijskim učinkovinam predstavlja pomemben javnozdravstveni problem, ena najbolj učinkovitih strategij pri boju proti vztrajnemu širjenju bakterijske rezistence pa je razvoj novih protibakterijskih učinkovin. Med najbolj pomembne tarče sodijo transpeptidazni encimi, ki sodelujejo pri prečnem povezovanju peptidoglikana in s tem zagotavljajo stabilnost bakterijske celične stene. Namen magistrske naloge je bil izraziti in izolirati tri transpeptidazne encime, vključene v biosintezo peptidoglikana, in sicer L,D-transpeptidazo B (LdtB) iz Escherichia coli, penicilin vezoči protein 1b (PBP1b) iz Streptococcus pneumoniae ter penicilin vezoči protein 3 (PBP3) iz Escherichia coli. Za izražanje rekombinantnih proteinov smo uporabili ekspresijski sev E. coli NiCo21 (DE3). Kompetentne celice smo transformirali s plazmidi z metodo toplotnega šoka, uspešnost transformacije pa smo potrdili s selekcijskimi gojišči z antibiotikom. Izolacijo proteinov smo izvedli z afinitetno kromatografijo, dodatno čiščenje pa s kromatografijo z ločevanjem po velikosti. Čistost proteinov smo ovrednotili z metodo SDS-PAGE, njihovo aktivnost pa preverili z ustreznimi fluorescenčnimi in vezavnimi testi. V okviru magistrske naloge smo uspešno izrazili in izolirali 99,3 mg encima LdtB ter potrdili njegovo encimsko aktivnost s fluorescenčnim testom aktivnosti. Uspešno smo izrazili in izolirali tudi PBP1b, vendar pri testiranju proteina nismo uspeli potrditi pričakovane biološke aktivnosti. Pri izražanju PBP3 smo s sekvenciranjem po Sangerjevi metodi potrdili prisotnost ustreznega genskega zapisa v plazmidu in izrazili manjše količine proteina, vendar zaradi nizkega izkoristka nismo uspeli pridobiti zadostne količine ustrezno čistega PBP3. LdtB, ki smo ga uspešno izrazili in izolirali tekom magistrske naloge, bo predstavljal osnovo za nadaljnje testiranje potencialnih novih protibakterijskih učinkovin na Katedri za farmacevtsko kemijo Fakultete za farmacijo Univerze v Ljubljani.

• Jezik: Slovenski jezik
• Ključne besede: transpeptidaze, peptidoglikan, bakterijska odpornost, rekombinantni proteini, afinitetna kromatografija, LdtB, PBP1b, PBP3
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Založnik: [K. Mesarič]
• Leto izida: 2026
• PID: 20.500.12556/RUL-183665
• UDK: 615.281:577.15
• COBISS.SI-ID: 282177539
• Datum objave v RUL: 17.06.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Expression and isolation of transpeptidase enzymes involved in peptidoglycan biosynthesis

Izvleček

Bacterial resistance to antibacterial agents represents a major public health problem and one of the most effective strategies in combating the persistent spread of bacterial resistance is the development of new antibacterial compounds. Among the most important targets are transpeptidase enzymes, which participate in the cross-linking of peptidoglycan and thereby ensure the stability of the bacterial cell wall. The goal of this master’s thesis was to express and isolate three transpeptidase enzymes involved in peptidoglycan biosynthesis, namely L,D-transpeptidase B (LdtB) from Escherichia coli, penicillin-binding protein 1b (PBP1b) from Streptococcus pneumoniae, and penicillin-binding protein 3 (PBP3) from Escherichia coli. The E. coli NiCo21 (DE3) expression strain was used for the expression of recombinant proteins. Competent cells were transformed with plasmids using the heat-shock method, and the success of the transformation was confirmed using a selective medium. Protein isolation was performed by affinity chromatography, followed by further purification using size-exclusion chromatography. Protein purity was evaluated by SDS-PAGE, while activity of the proteins was assessed using appropriate fluorescence-based and binding assays. Within the scope of this master’s thesis, we successfully expressed and isolated 99,3 mg of the enzyme LdtB and confirmed its enzymatic activity with a fluorescence-based activity assay. PBP1b was also successfully expressed and isolated; however, the expected biological activity could not be confirmed during testing. In the case of PBP3, the presence of the appropriate gene sequence in the plasmid was confirmed using Sanger sequencing and we managed to express a small amount of protein, but due to low yield and insufficient purity, it was not possible to obtain enough adequately pure PBP3. The successfully produced LdtB enzyme will serve as a basis for further testing of potential new antibacterial compounds at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana.

• Ključne besede: transpeptidases, peptidoglycan, bacterial resistance, recombinant proteins, affinity chromatography, LdtB, PBP1b, PBP3