title: Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.
abstract: Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran-Qom Express Way, Tehran, Iran.
authors.forename: Roghayyeh
authors.initials: R
authors.lastname: Baghban
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran-Qom Express Way, Tehran, Iran.
authors.forename: Seyed Latif Mousavi
authors.initials: SL
authors.lastname: Gargari
authors.affiliation: Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Rajabibazl
authors.affiliation: Department of Biological Sciences, Faculty of Sciences, Imam Hosein University, Tehran, Iran.
authors.forename: Shahram
authors.initials: S
authors.lastname: Nazarian
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran-Qom Express Way, Tehran, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Bakherad
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001905:Botulinum Toxins / Q000276:immunology*; D002162:Camelus / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000187:drug effects* / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D049329:Nanostructures / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D061905:Single-Domain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D001905:Botulinum Toxins; C096326:botulinum toxin type E
keywords: Pichia pastoris; botulinum neurotoxin E; nanobody