Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression of Sortase, a Transpeptidase for Cell Wall Sorting Reaction, from Staphylococcus aureus ATCC 6538p in Escherichia coli

  • LEE, KI-YOUNG (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • DONG-SUN SHIN (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • JUNG-MIN YOON (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • HEONJOONG KANG (MBL, School of Earch and Environmental Sciences, Seoul National University) ;
  • KI-BONG OH (Natural Products Research Institute, College of Pharmacy, Seoul National University, Graduate School of Agricultural Biotechnology, Seoul National Uni)
  • Published : 2002.06.01
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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 Cram-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 call be used in the search for sortase inhibitors In a short period of time.

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