These authors contributed equally to this article.
Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system
Article first published online: 18 JAN 2012
Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.
Biotechnology and Applied Biochemistry
Volume 59, Issue 1, pages 15–21, January/February 2012
How to Cite
Ezzine, A., M'Hirsi el Adab, S., Bouhaouala-Zahar, B., Hmila, I., Baciou, L. and Marzouki, M. N. (2012), Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system. Biotechnology and Applied Biochemistry, 59: 15–21. doi: 10.1002/bab.67
- Issue published online: 14 FEB 2012
- Article first published online: 18 JAN 2012
- Manuscript Accepted: 6 DEC 2011
- Manuscript Received: 22 AUG 2011
- AahI′ toxin;
- heterologous protein;
- Pichia pastoris
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.