title: Expression and purification of a recombinant scFv towards the exotoxin of the pathogen, Burkholderia pseudomallei.
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 degrees 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 degrees 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 Top10F'. 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, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.
authors.affiliation: Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia.
authors.forename: Kue-Peng
authors.initials: KP
authors.lastname: Lim
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
authors.forename: Sheila
authors.initials: S
authors.lastname: Nathan
mesh_terms: D000907:Antibodies, Bacterial / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001435:Bacteriophages / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015153:Blotting, Western; D016957:Burkholderia pseudomallei / Q000276:immunology* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000821:virology; D005098:Exotoxins / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D008970:Molecular Weight; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D014764:Viral Proteins