Ca2+ -independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Hidehiko Hirakawa, Suguru Ishikawa, Teruyuki Nagamune
Biotechnology Journal 2015, 10 (9): 1487-92
A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca(2+) concentrations and in the presence of Ca(2+)-binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+)-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations.


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