title: Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli.
abstract: Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.
authors.affiliation: Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada B3H 3J5.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Aminian
authors.forename: Sheila
authors.initials: S
authors.lastname: Sivam
authors.forename: Chiang W
authors.initials: CW
authors.lastname: Lee
authors.forename: Scott A
authors.initials: SA
authors.lastname: Halperin
authors.forename: Song F
authors.initials: SF
authors.lastname: Lee
mesh_terms: D000818:Animals; D004167:Diphtheria Toxin / Q000096:biosynthesis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D007106:Immune Sera; D051379:Mice; D037342:Pertussis Toxin / Q000096:biosynthesis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification*; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D004167:Diphtheria Toxin; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C518593:pertussis toxin-diphtheria toxin-tetanus toxin fusion protein; D037342:Pertussis Toxin