^*Author for Correspondence.
Faculty of Science and Technology,
Universiti Kebangsaan Malaysia,
43600 UKM Bangi,
Selangor Darul Ehsan, Malaysia
Tel: 60-3-8921-3862
Fax: 60-3-8925-2698
E-mail: sheila@pkrisc.cc.ukm.my

Key words: affinity chromatography, Burkholderia pseudomallei, exotoxin, scFv

Abstract.
Burkholderia pseudomallei is the causative agent of melioidosis, a fulminating disease in South East Asia and northern Australia. B. pseudomallei is known to secrete various extracellular products related to its pathogenesis, such as lethal exotoxin, protease and hemolysin. We have attempted to purify the exotoxin as studies have shown it to exhibit necrotic and cytotoxic activities and inhibit cellular protein synthesis. Purification was performed by antibody mediated affinity chromatography using previously generated single chain variable fragments (scFv) towards partially purified B. pseudomallei exotoxin coupled to a diaminodipropylamine column. B. pseudomallei was grown in BHIB + 2% glycerol under static conditions at 37°C for 7 days and crude extract was subjected to the scFv-linked column to capture the exotoxin. SDS-PAGE analysis exhibited a purified protein migrating as a single band bearing a size of approximately 37kDa. N-terminal protein sequencing and further bioinformatics analysis showed that the peptide shared some similarity with a putative decapeptide of the Pseudomonas aeruginosa exotoxin A and is present in both chromosomes 1 and 2 of B. pseudomallei. This suggests that the protein obtained could be the 37 kDa subunit of the B. pseudomallei exotoxin. Selected assays demonstrated that the purified product had no hemolytic, proteolytic or tyrosine phosphatase activity. The purified protein can aid in understanding the role of this virulence factor and serve as a candidate for vaccine development to combat melioidosis.