Purification of a burkholderia pseudomallei antigen via antibody mediated affinity chromatography

Kue Peng Lim, Rahmah Mohamed, Mohammed Noor Embi, Sheila Nathan

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1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)71-78
Number of pages8
JournalAsia-Pacific Journal of Molecular Biology and Biotechnology
Volume13
Issue number2
Publication statusPublished - 2005

Fingerprint

Burkholderia pseudomallei
Antibody Affinity
Exotoxins
Affinity Chromatography
Antigens
Melioidosis
Proteins
Single-Chain Antibodies
Hemolysin Proteins
Chromosomes, Human, Pair 2
Far East
Chromosomes, Human, Pair 1
Protein Sequence Analysis
Virulence Factors
Computational Biology
Complex Mixtures
Phosphoric Monoester Hydrolases
Glycerol
Tyrosine
Polyacrylamide Gel Electrophoresis

Keywords

  • Affinity chromatography
  • Burkholderia pseudomallei
  • Exotoxin
  • ScFv

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

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title = "Purification of a burkholderia pseudomallei antigen via antibody mediated affinity chromatography",
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.",
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AU - Embi, Mohammed Noor

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N2 - 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.

AB - 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.

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