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

Aymen Ezzine, Sonia M'Hirsi El Adab, Balkiss Bouhaouala-Zahar, Issam Hmila, Laura Baciou, Mohamed Najib Marzouki

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)15-21
Number of pages7
JournalBiotechnology and Applied Biochemistry
Volume59
Issue number1
DOIs
Publication statusPublished - 1 Jan 2012
Externally publishedYes

Fingerprint

Single-Domain Antibodies
Scorpions
Pichia
alcohol oxidase
Antibodies
Yeast
Escherichia coli
Yeasts
Recombinant proteins
Immunosorbents
Immunoglobulin Fragments
Saccharomyces
Secretory Pathway
Fungi
Recombinant Proteins
Histidine
Disulfides
Culture Media
Labels
Assays

Keywords

  • AahI' toxin
  • heterologous protein
  • His-Tag
  • N-glycosylation
  • nanobody
  • Pichia pastoris

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Molecular Medicine
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Drug Discovery
  • Process Chemistry and Technology

Cite this

Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system. / Ezzine, Aymen; M'Hirsi El Adab, Sonia; Bouhaouala-Zahar, Balkiss; Hmila, Issam; Baciou, Laura; Marzouki, Mohamed Najib.

In: Biotechnology and Applied Biochemistry, Vol. 59, No. 1, 01.01.2012, p. 15-21.

Research output: Contribution to journalArticle

Ezzine, Aymen ; M'Hirsi El Adab, Sonia ; Bouhaouala-Zahar, Balkiss ; Hmila, Issam ; Baciou, Laura ; Marzouki, Mohamed Najib. / Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system. In: Biotechnology and Applied Biochemistry. 2012 ; Vol. 59, No. 1. pp. 15-21.
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