Preparation of recombinant protein BirA

Encim BirA iz E. coli seva K12 spada med biotin protein ligaze (BPL), ki katalizirajo pripenjanje biotina na tarčne proteine. Uporabljamo ga lahko za biotinilacijo proteinov ter služi kot alternativa biotinilaciji s kemijskimi reagenti. Pri kemijski biotinilaciji lahko pride do večkratne biotinilacije tarčne molekule na različnih aminokislinskih ostankih, z uporabo encima BirA pa biotinilacija poteče le na enem lizinskem aminokislinskem ostanku znotraj 15 ak dolgega zaporedja. To tarčno zaporedje (GLNDIFEAQKIEWHE) imenujemo AviTag in ga lahko z uporabo molekulskega kloniranja dodamo na N- ali C-konec tarčnega proteina ali znotraj izpostavljene zanke proteina. Z uporabo BirA in AviTag-a lahko specifično biotiniliramo tarčne proteine, ki jih lahko uporabimo v nadaljnjih eksperimentalnih metodah. Zaradi zelo močne vezave biotina s (strept)avidinom lahko biotinilirane tarčne proteine očistimo, multimeriziramo, lokaliziramo v celici ali imobiliziramo. Z eksperimentalnim delom diplomske naloge nam je uspelo pripraviti aktiven rekombinantni protein BirA, ki biotinilira tarčne proteine z AviTag-om. Najprej smo z molekulskim kloniranjem pripravili vektor, na podlagi katerega smo v celicah E. coli BL21[DE3] inducirano izražali rekombinantni protein His6-MBP-3C-BirA. V topni frakciji je bila zadostna količina izraženega proteina, zato smo za nadaljnje delo uporabili le topno frakcijo. Iz kompleksne mešanice topne frakcije smo z uporabo Ni-afinitetne kromatografije in gelske filtracije očistili rekombinantni protein od preostalih komponent. Nazadnje smo dokazali, da je pripravljen in očiščen rekombinantni protein tudi aktiven in katalizira biotinilacijo substrata, fuziranega z AviTag-om. Za substrat smo uporabili mišji katepsin S (mCTSS) z AviTag-om. Po inkubaciji substrata in encima v prisotnosti ustreznih reagentov smo z NaDS-PAGE elektroforezo in prenosom western na membrani zaznali prisotnost biotinilacije. S tem smo dokazali aktivnost pripravljenega encima BirA, ki ga bo v prihodnje mogoče uporabljati za biotinilacijo substratov z AviTag-om.The BirA enzyme from E. coli strain K12 is one of the biotin protein ligases (BPLs), which catalyze the attachment of biotin to target proteins. In addition to enzymatic biotinylation, the target molecules can also be biotinylated using the appropriate chemical reagents. In chemical biotinylation, biotinylation of the target molecule can occur at multiple amino acid residues, whereas by using the enzyme BirA, biotinylation occurs at a single lysine amino acid residue within a 15 amino acid long sequence. This target sequence (GLNDIFEAQKIEWHE), named AviTag, can be fused to N- or C-terminus or within an exposed loop of a target protein using molecular cloning. Using BirA and AviTag, we can specifically biotinylate target proteins that can be used in further methods. Due to the very strong binding of biotin to (strept)avidin, biotinylated target proteins can be purified, multimerized, localized inside the cell, or immobilized. With our experimental work, we prepared an active recombinant BirA protein, which can biotinylate target proteins with an inserted AviTag. First, using molecular cloning, we prepared a His6-MBP-3C-BirA containing vector, which we used for the induced expression of recombinant protein in E. coli BL21[DE3] cells. There was a sufficient amount of recombinant protein in the soluble fraction, therefore we used only the soluble fraction for further work. Using Ni-affinity chromatography and size-exclusion chromatography, we purified the recombinant protein from the remaining components of the soluble fraction. Finally, we demonstrated that the prepared and purified recombinant protein is also active and catalyzes the biotinylation of the substrate fused with AviTag. Mouse cathepsin S (mCTSS) with AviTag was used as substrate. After incubation of the substrate and the enzyme with appropriate reagents, we used SDS-PAGE electrophoresis followed by western blot analysis to confirm successful biotinylation. We proved the activity of the prepared recombinant enzyme BirA, which can be further used for the biotinylation of substrates fused with AviTag