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KMID : 0545120020120030530
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 3 p.530 ~ p.533
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Expression of Sortase, a Transpeptidase for Cell Wall Sorting Reaction, from Staphyloccus aureus ATCC 6538p in Escherichia coli
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LEE, KI-YOUNG
SHIN, DONG-SUN/YOON, JUNG-MIN/KANG, HEONJOONG/OH, KI-BONG
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Abstract
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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.
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