Kloniranje in izražanje proteina toplotnega šoka Grp94

Duša, Žiga (author), Pečar Fonović, Urša (mentor), Cingl, Jernej (comentor)

Proteini toplotnega šoka (Hsp) sodelujejo pri zvijanju mnogih celičnih proteinov in so posredno vpleteni v razvoj različnih bolezni. Družina Hsp90 ima številne t.i. proteine kliente, ki so se izkazali za onkogene. V endoplazemskem retikulumu se nahaja z glukozo reguliran protein, velik 94 kDa (Grp94), ki je ena od izooblik družine Hsp90. Selektivno zaviranje omenjene izooblike bi vplivalo na zvijanje nekaterih onkogenih proteinov brez vpliva na delovanje drugih izooblik Hsp90. Namen magistrske naloge je bil klonirati gen HSP90B1, izraziti človeški protein Grp94 ter določiti vezavno afiniteto dveh spojin do Grp94 – znanega neselektivnega zaviralca, ki deluje na C-končno domeno, novobiocina in spojine UL-Hsp90 1. Slednja je bila pripravljena na Fakulteti za farmacijo, Univerze v Ljubljani za namene zaviranja članov družine Hsp90 na C-končni domeni. Gen HSP90B1 smo vstavili v bakterijski ekspresijski vektor pET28. Po neuspešnem klasičnem molekulskem kloniranju smo z metodo sestavljanja po Gibsonu uspešno pripravili konstrukt pET28, ki je vseboval gen HSP90B1. Izražanje smo inducirali v sevu E. coli NiCo21 (DE3). Za uspešno biosintezo celotnega Grp94 je bila potrebna sočasna transformacija bakterij s plazmidom pRARE2, ki zagotavlja prenašalne ribonukleinske kisline za kodone, redke v izbranem bakterijskem sevu. Optimizirali smo tudi pogoje izražanja in določili, da za ekspresijo zadostuje 0,1 mM koncentracija izopropil-β-D-1 tiogalaktopiranozida v gojišču 2× YT in pri temperaturi izražanja 16 °C. Izolacija proteina je potekala v dveh korakih: najprej z nikelj – kelatno afinitetno kromatografijo in nato še s kromatografijo z ločevanjem po velikosti. Prisotnost proteina Grp94 smo potrdili z analizo s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecil sulfata in prenosom western, ter njegovo koncentracijo določili z metodo po Bradfordu. Izolirani človeški protein Grp94 smo nato uporabili za določanje vezavne afinitete izbranih spojin z metodo termoforeze na mikroskali. Na Grp94 smo določili vrednost konstante disociacije za novobiocin (217,5 ± 80,17 µM), medtem ko za spojino UL-Hsp90-1 z enako metodo vrednosti nismo uspeli določiti. To pomeni, da se spojina UL-Hsp90-1 na protein Grp94 verjetno ne veže.

• Language: Slovenian
• Keywords: rak, proteini toplotnega šoka, Grp94, izražanje v E. coli NiCo21 (DE3), termoforeza na mikroskali
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-173009
• Publication date in RUL: 12.09.2025

Secondary language

• Language: English
• Title: Cloning and expression of heath shock protein Grp94

Abstract

Heat shock proteins are involved in the folding of many cellular proteins and are indirectly implicated in the development of various diseases. The Hsp90 family contains numerous so called client proteins that have been identified as oncogenic drivers. In the endoplasmic reticulum, glucose regulated protein 94 (Grp94) is present as one of the isoforms of the Hsp90 family. Selective inhibition of this isoform could affect the folding of certain oncogenic proteins without interfering with the activity of other Hsp90 isoforms. The aim of this master’s thesis was to clone the gene HSP90B1, express human Grp94 and to determine the binding affinity of two compounds towards Grp94: non-selective C-terminal domain inhibitor novobiocin, and the compound UL-Hsp90-1. The latter was synthesized at the Faculty of Pharmacy, University of Ljubljana, for the purpose of inhibiting members of the Hsp90 family at the C-terminal domain. The HSP90B1 gene was inserted into the bacterial expression vector pET28. After unsuccessful attempts with classical molecular cloning, the construct pET28 containing the HSP90B1 gene was successfully prepared using Gibson assembly. Expression was induced in the E. coli strain NiCo21 (DE3). For successful biosynthesis of full-length Grp94, co transformation of bacteria with the plasmid pRARE2 was required, providing transfer ribonucleic acids for codons that are rare in the chosen bacterial strain. Expression conditions were further optimized and it was determined that induction with 0.1 mM isopropyl β-d-1 thiogalactopyranoside in 2× YT medium at 16 °C was sufficient for protein expression. Protein isolation was performed in two steps: first by metal-chelate affinity chromatography, followed by size-exclusion chromatography. The presence of Grp94 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis. Protein concentration was determined using the Bradford assay. The isolated human Grp94 was then used to assess the binding affinity of the selected compounds by microscale thermophoresis. A dissociation constant for novobiocin binding to Grp94 was successfully determined (217.5 ± 80.17 µM), while no binding constant could be determined for UL Hsp90-1 under the same conditions. This could indicate that UL-Hsp90-1 does not bind to Grp94.

• Keywords: cancer, heath shock proteins, Grp94, expression in E. coli NiCo21 (DE3), microscale thermophoresis