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Characterizing Sortase A Mutants with Altered Binding Selectivity
AuthorBolt, Alexander James
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Post-translational protein fusion has potential to meet the diverse and complex needs of many current problems in biotechnology. Traditional approaches such as genetic fusion are heavily constrained to simple protein fusions that can be expressed within the host cell. Enzymatic ligation mediated by Staphylococcus aureus sortase A (SrtA) broadens the scope of protein fusion by ligating proteins post-translationally, given that the target proteins bear the two substrates recognized by SrtA: short peptides of 3-5 amino acids in length. Nonetheless, one of these tags, polyglycine, is forcibly constrained to the protein N-terminus. Previous efforts have sought to overcome this limitation by engineering SrtA with substrate specificity for a motif found in pilin formation, known as pilin box, using directed evolution. In this work, engineered SrtA mutants are analyzed both on the yeast surface and in soluble form for altered expression levels or activity toward natural polyglycine or the pilin substrate. Expression levels in some mutants increased by over 100-fold. Mutants on yeast showed higher selectivity for pilin box, no longer recognizing polyglycine and maintaining pilin box specificity at a level comparable to eSrtA, an evolved SrtA with higher catalytic activity. A reliable, facile method is used for the quantification of SrtA-mediated ligation product yields using elastin-like polypeptide (ELP) purification and fluorescence assays. SrtA WT showed specificity for both GGG and P0, whereas mutants 1 and 8 were selective toward P0 and GGG, respectively. These findings provide new approaches to characterize transpeptidases with more than one substrate on the yeast surface or in solution, and pave the way for future site-specific protein ligation tools to generate post-translational protein fusions.