title: Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin.
abstract: The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.
authors.affiliation: Centre for Gene Analysis and Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D.E., Malaysia.
authors.forename: Yu-Ching
authors.initials: YC
authors.lastname: Su
authors.forename: Kue-Peng
authors.initials: KP
authors.lastname: Lim
authors.forename: Sheila
authors.initials: S
authors.lastname: Nathan
mesh_terms: D016957:Burkholderia pseudomallei / Q000276:immunology*; D003001:Cloning, Molecular; D005098:Exotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D008554:Melioidosis / Q000276:immunology
chemical_list: D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region