Purification and functional analysis of the recombinant protein isolated from E. coli by employing three different methods of bacterial lysis

In this paper, the purification of the human recombinant protein expressed in E. coli using the GSTGene Fusion System, by applying various methods of bacterial lysis: sonication, freeze/thaw and beadbeating, is presented. The study was an attempt to compare the properties of the proteins obtained by the sonication method, recommended by manufacturers but inaccessible for many researchers, with those obtained using two other readily available lysis methods. The data show that all purified proteins were soluble and intact with the highest protein yield being obtained via the freeze/thaw method. The results of functional analysis indicate that the proteins purified using the sonication and freeze/thaw methods of lysis exhibited similar DNA binding affinity, while the protein purified by beadbeating was also functional but with a lower binding affinity. The conclusion of this study is that all three lysis methods could be successfully employed for protein purification. U ovom radu prikazano je preciscavanje humanog rekombinantnog proteina eksprimiranog u E. coli. Rekombinantni protein, fuzionisan sa glutation-S-transferaznim domenom (GST), izolovan je primenom tri razlicite metode bakterijske lize: sonifikovanje, zamrzavanje/odmrzavanje i razbijanje bakterija kuglicama. Proizvodjac GST fuzionog sistema preporucuje bakterijsku lizu sonifikovanjem, ali ova metoda je cesto nedostupna istrazivacima jer zahteva skupu opremu i dugotrajnu optimizaciju uslova. Cilj naseg rada je bio da utvrdimo da li se koriscenjem i druge dve metode, lako primenljive u svakoj laboratoriji, mogu dobiti proteini odgovaraju- cih karakteristika. Nasi rezultati pokazuju da sve tri metode lize omogucavaju izolovanje solubilnih i intaktnih proteina, a da je najveci prinos dobijen primenom metode zamrzavanja/odmrzavanja bakterija. Na osnovu funkcionalne analize zakljucili smo da proteini dobijeni metodom sonifikovanja i ponovljenog zamrzavanja/odmrzavanja pokazuju visok afinitet za specificno vezivanje za DNK. Rekombinantni protein dobijen primenom metode razbijanja bakterija kuglicama pokazuje smanjen afinitet za vezivanje za DNK, ali se, takodje, moze koristiti u analizama interakcija proteina i DNK. Zakljucak nasih istrazivanja je da se sve tri metode lize bakterija mogu uspesno primeniti za izolovanje rekombinantnih proteina.