Expression of Sortase, a Transpeptidase for Cell Wall Sorting Reaction, from Staphyloccus aureus ATCC 6538p in Escherichia coli

다국어 초록 (Multilingual Abstract)

This paper describes the development of an enzymatic assay system for the identification of specific inhibitors of sortase, a transpeptidase that cleaves surface proteins of Gram-positive bacteria, from Staphylococcus aureus ATCC 6538p for antibacterial drug discovery. The coding region of the enzyme was amplified with the exception of the N-terminal membrane anchor sequence, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The enzyme activity was determined by quantifying increased fluorescence intensity upon cleavage of synthetic Dabcyl-QALPETGEE-Edans peptide. The results suggest that the developed in vitro assay system can be used in the search for sortase inhibitors in a short period of time.