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Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system

Authors

  • Aymen Ezzine,

    Corresponding author
    1. Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia
    • Specialist in Biochemistry and Molecular Biology/Postdoctoral Researcher, Laboratoire de Chimie Physique, Bât 350 CNRS UMR 8000 Université Paris 11, 91405 Orsay Cedex, France. Tel. +33 (0)1 69 15 75 65; web: http://www.lcp.u-psud.fr
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  • Sonia M'Hirsi el Adab,

    1. Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia
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  • Balkiss Bouhaouala-Zahar,

    1. Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunis, Tunisia
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  • Issam Hmila,

    1. Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunis, Tunisia
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  • Laura Baciou,

    1. Laboratoire de Chimie Physique, Université Paris Sud 11, Orsay, France
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    • These authors contributed equally to this article.

  • Mohamed Najib Marzouki

    1. Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia
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    • These authors contributed equally to this article.


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.

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