Molecular cloning and preparation of recombinant Cas9 and ExoIII protein variants in Escherichia coli

Priprava in izolacija rekombinantnih proteinov je eden od najpogostejših postopkov v raziskavah na področju ved o življenju. Kljub njegovi široki razširjenosti pa vsak protein zahteva individualizirano izvedbo fermentacije in izolacije, ki jo določamo preko sistematičnega testiranja pogojev gojenja bakterijskih celic pred in po indukciji izražanja proteina. Ti pogoji so odvisni od lastnosti posameznega proteina. Protein Cas9 predstavlja eno od ključnih komponent sistema CRISPR/Cas9, ki se v zadnjem času vse bolj uveljavlja kot orodje za urejanje genoma. Njegova povezava z eksonukleazo III (ExoIII) predstavlja možnost za izboljšanje sistema, in sicer preko povečanja vrzeli na mestu dvojnega preloma, ki ga povzroči Cas9. Na ta način pride do izbrisa večjega dela gena in posledično večje uspešnosti njegovega izbitja (ang. knock-out). V okviru diplomske naloge smo pripravili nukleotidna zaporedja z zapisom za rekombinantna proteina Cas9 in ExoIII z dodanim zapisom za α-vijačnico na 3\u27 koncu genskega konstrukta. Uporabili smo dva para ortogonalnih α-vijačnic. Te se ob vnosu obeh izoliranih proteinov v celico združijo v ovito vijačnico in na ta način povežejo proteina. Zapise za proteine smo vstavili v ekspresijski vektor pET-28b-Cas9-His ter z njimi transformirali ustrezne ekspresijske seve E. coli. Po fermentaciji smo proteine preko histidinske oznake izolirali z nikljevo afinitetno kromatografijo ter jih skoncentrirali. Tako pripravljeni vzorci so primerni za testiranje učinkovitosti povezave Cas9 – ExoIII ter potencialno uporabo v poskusih urejanja genoma.Preparation and isolation of recombinant proteins is one of the most common procedures in life sciences\u27 research. Because each protein requires different conditions for fermentation and specific isolation technique, there is no universal protocol to follow. We usually determine these optimal conditions, which depend on the characteristics of the expressed protein, by a series of testing fermentations. The Cas9 protein is one of the key elements of the CRISPR/Cas9 system, which is a promising tool in genome editing. One of the possible improvements of this technique is connecting Cas9 with exonuclease III (ExoIII), which is capable of forming a bigger gap at the site of double strand break. By doing so a greater part of the target gene is deleted and the frequency of knock-out increases. We prepared the nucleotide sequences encoding recombinant proteins Cas9 and ExoIII in fusion with two pairs of orthogonal α helices, which form a coiled coil when simultaneously present in vitro or in vivo. By doing so they physically connect the two recombinant proteins. We inserted our constructs into the expression vector pET-28b-Cas9-His and transformed them into E. coli expression strains. We isolated the proteins, tagged with a hexahistidine group, with Ni2+-NTA affinity chromatography and concentrated the samples. These isolated and purified recombinant proteins can be further used for testing the efficiency of Cas9 – ExoIII connection and for genome editing experiments