Optimizacija izražanja in čiščenja rekombinantne glikoziltransferaze O-β-N-acetilglukozamin transferaze

Pavšič, Selena (avtor), Bratkovič, Tomaž (mentor)

Protein O GlcNAc transferaza OGT je edini encim, ki katalizira pripenjanje beta N acetilglukozamina na hidroksilno skupino aminokislinskih ostankov serina in treonina znotrajceličnih proteinov. N acetilglukozaminilacija je obsežna dinamična posttranslacijska modifikacija, ki se močno prepleta s signalnimi potmi fosforilacije proteinov. Modificirani proteini so vključeni v mnoge celične procese, motnje v njihovi homeostazi pa so povezane s številnimi kroničnimi boleznimi. Zaradi biološkega pomena OGT predstavljajo zaviralci ključno orodje za razumevanje mehanizmov modifikacije, odkrivanje njenih neznanih vlog ter validiranje OGT kot terapevtske tarče. Učinkovita proizvodnja funkcionalnih proteinov v heterolognih gostiteljskih sistemih je eden izmed glavnih temeljev sodobne biotehnologije. V magistrski nalogi smo se posvetili optimizaciji izražanja in čiščenja rekombinantnega proteina OGT. Namen naloge je bil pridobiti aktiven in kromatografsko čist protein ustrezne koncentracije z uporabo ekspresijskega sistema E. coli. V prvem delu naloge smo protein testno izražali pri različnih koncentracijah induktorja v treh bakterijskih sevih NiCo21DE3, Rosetta gami2DE3pLysS in SHuffle T7 Express lysY. Za izboljšanje izražanja smo ekspresijska sistema NiCo21DE3 ter SHuffle T7 Express lysY transformirali s plazmidom pRARE2, ki vsebuje sedem tRNA za prepoznavo redkih kodonov. Analiza celičnih lizatov s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata je pokazala, da ima OGT najvišjo stopnjo izražanja v bakterijskem sevu NiCo21DE3 z vstavljenim pRARE2 pri koncentraciji induktorja 0,2 mM. Drugi del magistrske naloge zajema večstopenjsko čiščenje proteina, pridobljenega z izražanjem v povečanem obsegu. Preskušali smo vplive načina celične lize na ustrezno strukturo proteina, način odstranjevanja nativnih proteinov E. coli ter tehnike koncentriranja proteina za dosego želene količine in čistote. Za najuspešnejše se je izkazala liza bakterijskih celic s soniciranjem, kombinacija izolacije in čiščenja s kovinsko kelatno kromatografijo in gelsko filtracijo ter končno koncentriranje z ultrafiltracijo. V vse pufre smo dodali 0,1 odstotek Tweena 20. Doseženo ustrezno koncentracijo smo potrdili spektrofotometrično, čistoto z vizualizacijo lise pravilne velikosti na poliakrilamidnem gelu, aktivnost pa s kvantitativnim encimskim testom na osnovi fluorescence.

• Jezik: Slovenski jezik
• Ključne besede: OGT, redki kodoni, optimizacija, izražanje, čiščenje
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-129670
• Datum objave v RUL: 07.09.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Expression and purification optimization of recombinant O-linked β-N-acetylglucosamine transferase

Izvleček

Protein O GlcNAc transferase OGT is the sole enzyme to catalyse the attachment of beta N acetylglucosamine to the hydroxyl group of serine and threonine residues of intracellular proteins. N acetylglucosamination is an abundant dynamic post translational modification that shows extensive crosstalk with protein phosphorylation. Modified proteins are involved in a number of important cell processes and the disruption of homeostasis has been reported to be associated with many human diseases. Considering its biological importance, OGT inhibitors are a convenient tool for understanding the mechanisms of modification, discovering its unknown functions and validating OGT as a therapeutic target. The efficient production of functional proteins in heterologous host systems plays a crucial role in modern biotechnology. In the master s thesis, we focused on the optimization of OGT expression and purification. Our aim was to isolate an active and chromatographically pure protein of optimal concentration using the E. coli expression system. In the first part of the task we screened the expression at different inducer concentrations in three bacterial strains NiCo21DE3, Rosetta gami2DE3 pLysS and SHuffle T7 Express lysY. To improve protein expression, NiCo21DE3 and SHuffle T7 Express lysY expression systems were transformed with the plasmid pRARE2 harboring seven rare codon tRNAs. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that OGT had the highest expression levels in the bacterial strain NiCo21DE3 transformed with pRARE2 and in the presence of 0.2 mM inducer. The second part represented a multistep purification of the protein obtained by optimized scale up expression. We probed the effects of the cell lysis methods on the correct protein structure, the removal of native E. coli proteins and techniques for concentrating proteins to achieve the desired amount and purity. We discovered that the purification by immobilized metal affinity chromatography followed by size exclusion chromatography led to the recombinant product of highest purity. Prior to that, bacterial cells were lysed by sonication. The recombinant enzyme was concentrated by ultrafiltration. All buffers were supplemented with 0.1 percent Tween 20. Finally, the concentration of protein was determined spectrophotometrically, and its purity was confirmed by polyacrilamide gel visualization. Enzyme s activity was assessed by a direct fluorescent activity assay.

• Ključne besede: OGT, rare codons, optimization, expression, purification