Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

William Barton


In the past two decades, the field of protein engineering has evolved rapidly to include new genetic and chemical techniques to alter protein function. Protein engineering seeks to improve enzyme properties through powerful methods that specifically incorporate novel or improved function in proteins. One such method is protein ligation, which is used to selectively link synthetic and recombinant polypeptides. Due to the limitations of current protein labeling techniques, simple site-specific modification methods remain in high demand. Use of enzyme-based labeling has been the focus of various studies because of its substrate specificity. Sortase-mediated transpeptidation is one approach that has been well documented. Staphylococcus aureus sortase A (SrtAstaph), a membrane-anchored cysteine transpeptidase present in gram-positive bacteria, covalently anchors virulence-associated surface proteins to the peptidoglycan cross bridge of the cell wall. SrtAstaph, one of the most characterized sortases, has found numerous applications in the semi-synthesis of protein and peptide conjugates. While current studies have demonstrated the growing range of applications for sortase A, the enzyme itself has seen very few improvements. In steady-state kinetic analysis, the calculated K cat value of SrtAstaph was 2.27 × 10−5 s−1 indicative of its slow in-vitro turnover rate. Due to sortase’s relative inefficiency, several studies documented the use of excessive amounts of the enzyme in vitro (>30μM) or reactions were incubated for long periods. Through the use of directed evolution, we aimed to improve the catalytic activity of sortase A. Using random mutagenesis and an in vivo bacterial-based screen we isolated a variant that showed a 13-fold increase in its catalytic efficiency when compared to wild-type. This sortase mutant will enable more efficient labeling of LPETG-tagged substrates and will provide further insight into the enzyme’s molecular mechanism of catalysis, which is currently limited.


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