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Efficient expression of the anti‐ Aah I' scorpion toxin nanobody under a new functional form in a Pichia pastoris system
Author(s) -
Ezzine Aymen,
M'Hirsi el Adab Sonia,
BouhaoualaZahar Balkiss,
Hmila Issam,
Baciou Laura,
Marzouki Mohamed Najib
Publication year - 2012
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.67
Subject(s) - pichia pastoris , recombinant dna , pichia , biology , escherichia coli , alcohol oxidase , heterologous expression , biochemistry , yeast , microbiology and biotechnology , heterologous , saccharomyces cerevisiae , gene
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 Aah I′ 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.