สถาบันวิจัยวิทยาศาสตร์สาธารณสุข

National Institute of Health of Thailand

Authors : Wimol Petkanchanapong *,***, Piacham T*, Prachayasittikul V*, Bulow L **


Affiliations : 
      * Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand

** Department of Pure and Applied Biochemistry, Center of Chemistry and Chemical Engineering, Lund University, Lund, Sweden
                        *** National Institute of Health, Department of Medical Science, Thailand
 
 
Source:             Presented at Exhibition of Graduate Thesis II: 2001. Mahidol University, Bangkok, Thailand
 
 
Language:         English
 
 
Abstract:
 
Burkholderia pseudomallei  is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. Protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We used phage display technology to identify peptides binding to B. pseudomallei  protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys peptide was decoded and subjected to gene fusion and protein engineering to derive the chimeric autofluorescence protein having the protease binding site and green fluorescent protein molecule. The chimeric protease binding-green fluorescent protein (PB-GFP) was successfully constructed. It bound to the B. pseudomallei  protease while exhibited flu rescence emission of GFP. The lower limit of detection was 250 ng of protease. It provided no binding activity to subtilisin, trypsin, proteinase K and RNase. A simple one step protocol for detection of B. pseudomallei  using chimeric protein PB-GFP is now ongoing.