Primerjava dveh bakterijskih ekspresijskih sistemov za pripravo mišjega katepsina L

Zajec Hudnik, Tina (avtor), Turk, Boris (mentor)

Bakterijski ekspresijski sistemi, kot je E. coli, so zaradi cenovne dostopnosti, preprostosti, kratkega generacijskega časa organizma in dobro poznane genetike priljubljena izbira pri pripravi rekombinantnih proteinov. Priprava rekombinantnih proteinov v E. coli pa ima tudi določene omejitve, na primer nezmožnost izvajanja posttranslacijskih modifikacij in oteženo zvijanje heterolognih rekombinantnih proteinov, kar pogosto vodi v njihovo agregacijo. Omejitve bakterijskih ekspresijskih sistemov lahko privedejo do neuspešne priprave predvsem evkariontskih proteinov, kar rešujemo z ustreznimi prilagoditvami parametrov priprave. Prilagoditvi, ki nam pogosto omogočata doseganje večjih izkoristkov priprave, sta uporaba specializiranih sevov in izražanje s koekspresijo šaperonov. Tvorba disulfidnih vezi je pri vzpostavljanju pravilne proteinske strukture pogosto stopnja, ki omejuje hitrost, zato smo se v sklopu diplomskega dela odločili primerjati dva bakterijska ekspresijska sistema, ki omogočata lažjo tvorbo disulfidnih vezi v citoplazmi bakterij. V specializiranem sevu Rosetta-gami 2 (DE3) in v klasičnem ekspresijskem sevu BL21 (DE3) pLysS smo pripravili mišji katepsin L, ki v nativni strukturi vsebuje tri disulfidne vezi. Za pripravo v sevu BL21 (DE3) smo uporabili sistem CyDisCo, ki prek koekspresije šaperonov omogoča lažje vzpostavljanje disulfidnih vezi v reducirajočem okolju citoplazme bakterij. Eksperiment smo izvajali v štirih paralelkah, saj nas je poleg vpliva bakterijskega ekspresijskega sistema zanimalo tudi, kako izbira gojišča LB oziroma TB vpliva na uspešnost priprave mišjega prokatepsina L (mpkatL) v nativni obliki. Zapis za proobliko mišjega katepsina L smo s tehniko IVA vstavili v izbran ekspresijski vektor in pripravili protein v obeh izbranih sistemih. Poleg vpliva ekspresijskega sistema smo preverili tudi, kako izbira gojišča vpliva na pripravo izbranega proteina v obeh ekspresijskih sistemih. Pripravljeni mišji prokatepsin L smo izolirali z nikljevo afinitetno kromatografijo, aktivirali in s titracijo aktivnega mesta določili njegovo aktivno koncentracijo. V obeh ekspresijskih sistemih smo uspešno pripravili aktiven mišji katepsin L (mkatL) z nekoliko večjim izkoristkom priprav pri kombinacijah seva Rosetta-gami 2 (DE3) in gojišča LB ter sistema CyDisCo in gojišča TB. Glede na predstavljene rezultate je za nadaljnjo optimizacijo pogojev priprave mišjega katepsina L najbolje uporabiti omenjeni kombinaciji ekspresijskega sistema in gojišča.

• Jezik: Slovenski jezik
• Ključne besede: mišji katepsin L, CyDisCo, Rosetta-gami, TB, LB
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-149279
• COBISS.SI-ID: 167350019
• Datum objave v RUL: 06.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Comparison of Two Bacterial Expression Sytems for the Production of Mouse Cathepsin L

Izvleček

Bacterial systems such as E. coli are a popular choice for the production of recombinant proteins due to their affordability, simplicity, short generation time of the organism, and well-studied genetics. However, expressing proteins in E. coli also has certain limitations such as the inability to perform post-translational modifications and protein aggregation tendency of heterologous proteins. These hindrances can often lead to unsuccessful preparation of eukaryotic proteins, which can be resolved by appropriate adjustments of preparation parameters. Two common adaptations that often enable higher yields of preparation are the use of specialized strains and co-expression with chaperones. Since the formation of disulfide bonds in establishing the correct protein structure is often a rate-limiting step, we decided to compare two bacterial systems, which enable disulfide bond formation in the cytoplasm of bacteria. Therefore, we prepared mouse procathepsin L, a protein that contains three disulfide bonds in its native structure, in a specialized strain called Rosetta-gami 2 (DE3) and in a classical expression strain BL21 (DE3) pLysS. In the BL21 (DE3) strain, we employed the CyDisCo system, which facilitates the establishment of disulfide bonds in the reducing environment of the bacterial cytoplasm through co-expression with chaperones. The experiment was conducted in four replicates, as we were interested not only in the influence of the bacterial strain but also in how the presence of either LB or TB growth medium affects the success of preparing mouse procathepsin L in its native form. Using the IVA technique, we inserted the gene for mouse procathepsin L into the selected expression vector and prepared the protein in both selected systems. Besides investigating the impact of the expression system, we also examined how the choice of growth media affected the preparation of the selected protein in both expression systems. The prepared mouse procathepsin L was isolated using nickel affinity chromatography and activated. Afterwards, its active concentration was determined with titration of the active site. In both expression systems, we managed to successfully prepare active mouse cathepsin L with slightly higher preparation yields in the combination of the Rosetta-gami 2 (DE3) strain with LB growth medium and the CyDisCo system with TB growth medium. Based on the presented results, it is best to use these specific combinations of the expression system and growth media for further optimization of mouse cathepsin L preparation conditions.

• Ključne besede: mouse cathepsin L, CyDisCo, Rosetta-gami, LB, TB