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Sortasesare enzymes that are responsible for the attachment ofsecreted proteins to the cell wall of Gram-positive bacteria. Hereby,the sortases recognize short, five-residue amino acid sequences presentin the target proteins and fuse them to the peptidoglycan layer viaa transpeptidation reaction, creating a new peptide bond between theC-terminus of the recognition sequence and the cell wall. The transpeptidationactivity of sortases is widely used in protein engineering for modificationof target proteins. The majority of protocols rely on the high activityof the well-characterized Staphylococcus aureus SrtAand variants thereof, while sortases from other classes are not usedfor this purpose. This can be attributed to the lower activity ofother sortases and to the limited sequence specificity data availablefor the different sortases. We set out to determine the sequence specificityof Bacillus anthracis SrtB. To this end, we developeda new method for sequence specificity determination of sortases orother bond-forming enzymes that recognize an amino acid sequence.Using mixtures of recognition peptides of limited complexity, whichare reacted with biotinylated substrates, the biotinylated transpeptidationproducts are isolated with magnetic streptavidin beads and analyzedvia liquid chromatography and mass spectrometry. With this, peptidesequences that are recognized by the sortase and function as substratescan be determined and quantified. The method, developed with the highlyactive evolved SrtA from S. aureus, allowed for thefirst time unbiased in-depth analysis of the sequence specificityfor SrtB from B. anthracis, which is 104-fold less active than SrtA from S. aureus.
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