Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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Expression and Purification of a Recombinant scFv towards the Exotoxin of the Pathogen, Burkholderia pseudomallei

  • Lim, Kue-Peng (Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia) ;
  • Li, Hong-Bin (Department of Immunology, The Scripps Research Institut) ;
  • Sheila Nathan (Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia)
  • Published : 2004.06.01
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Abstract

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30$^{\circ}C$ until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30$^{\circ}C$ for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Topl0F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, com-pared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

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