Oligomeric proteins are proteins composed of multiple polypeptide chains. Among enzymes, homodimers are predominant, i.e., dimers where both subunits are identical. By cross-linking the protein covalently, we can improve its stability, increase specificity and complexity of reactions, and achieve allosteric and cooperative effects that enhance reaction rates. Non-specific oligomerization of proteins can be achieved in various ways, most commonly by covalently linking proteins with cross-linkers.
The goal of this thesis was to covalently link xylelain, an extracellular cysteine protease originating from the bacterium Xylella fastidiosa.
Recombinant xylelain, with an added tyrosine amino acid residue and an added cysteine amino acid residue, expressed and isolated from E. coli, was covalently linked using tyrosinase. It is an enzyme that oxidizes tyrosine to o-quinone, which then reacts with the thiol group of the cysteine amino acid residue. Despite our optimization efforts, covalent linking of xylelain using tyrosinase was not effective, and we were also unable to isolate the xylelain dimer. Therefore, we additionally covalently linked xylelain using SnoopLigase, where we used this enzyme to link xylelain with the SnoopTagJr and DogTag labels.
To determine whether the proximity of the two xylelain subunits has allosteric effects, we measured the effect of heparin on the activity of the obtained xylelain dimers. The xylelain dimer covalently linked with SnoopLigase was not active, so we measured the dependence of xylelain dimer activity on heparin concentration in a mixture of the dimer covalently linked with tyrosinase and unreacted xylelain. We measured that the K$_{0,5}^h$ of heparin is 0.337 µg/mL, and the F$_\infty$ of the mixture is 36.9%.
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