title: Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system.
abstract: Most large-scale microbial production of recombinant proteins are based on Escherichia coli, yeasts, or filamentous fungi systems. Using eukaryotic hosts, antibody fragments are generally expressed by targeting to the secretory pathway. This enables not only efficient disulfide bond formation but also secretion of soluble and correctly folded product. For this goal, a recombinant vector was constructed to produce a single-domain antibody (NbAahI'22) directed against AahI' scorpion toxin using the methylotrophic yeast Pichia pastoris. The corresponding complementary DNA was cloned under control of the alcohol oxidase promoter in frame with the Saccharomyces α-factor secretion signal and then transferred to P. pastoris cell strain X-33. Using Western blot, we detected the expression of the recombinant NbAahI'22 exclusively in the culture medium. Targeting to the histidine label, the secreted nanobody was easily purified on nickel-nitrilotriacetic acid resin and then tested in enzyme-linked immunosorbent assay. Interestingly, the production level of the NbAahI'22 in its new glycosylated form reached more than sixfold that obtained in E. coli. These findings give more evidence for the utilization of P. pastoris as a heterologous expression system.
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia. e.aymen@yahoo.fr
authors.forename: Aymen
authors.initials: A
authors.lastname: Ezzine
authors.forename: Sonia
authors.initials: S
authors.lastname: M'hirsi El Adab
authors.forename: Balkiss
authors.initials: B
authors.lastname: Bouhaouala-Zahar
authors.forename: Issam
authors.initials: I
authors.lastname: Hmila
authors.forename: Laura
authors.initials: L
authors.lastname: Baciou
authors.forename: Mohamed Najib
authors.initials: MN
authors.lastname: Marzouki
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012605:Scorpions / Q000235:genetics*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins