Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system.
Identifieur interne : 000066 ( Ncbi/Curation ); précédent : 000065; suivant : 000067Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system.
Auteurs : Aymen Ezzine [Tunisie] ; Sonia M'Hirsi El Adab ; Balkiss Bouhaouala-Zahar ; Issam Hmila ; Laura Baciou ; Mohamed Najib MarzoukiSource :
- Biotechnology and applied biochemistry [ 1470-8744 ]
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Antibodies, Recombinant Proteins.
- chemical , isolation & purification : Antibodies, Recombinant Proteins.
- chemical , metabolism : Antibodies, Recombinant Proteins.
- genetics : Pichia, Scorpions.
- metabolism : Pichia.
- Animals, Gene Expression.
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.
DOI: 10.1002/bab.67
PubMed: 22332740
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Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia. e.aymen@yahoo.fr</nlm:affiliation><country xml:lang="fr">Tunisie</country><wicri:regionArea>Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis</wicri:regionArea><placeName><settlement type="city">Tunis</settlement><region nuts="2">Gouvernorat de Tunis</region></placeName></affiliation></author><author><name sortKey="M Hirsi El Adab, Sonia" sort="M Hirsi El Adab, Sonia" uniqKey="M Hirsi El Adab S" first="Sonia" last="M'Hirsi El Adab">Sonia M'Hirsi El Adab</name></author><author><name sortKey="Bouhaouala Zahar, Balkiss" sort="Bouhaouala Zahar, Balkiss" uniqKey="Bouhaouala Zahar B" first="Balkiss" last="Bouhaouala-Zahar">Balkiss Bouhaouala-Zahar</name></author><author><name sortKey="Hmila, Issam" sort="Hmila, Issam" uniqKey="Hmila I" first="Issam" last="Hmila">Issam Hmila</name></author><author><name sortKey="Baciou, Laura" sort="Baciou, Laura" uniqKey="Baciou L" first="Laura" last="Baciou">Laura Baciou</name></author><author><name sortKey="Marzouki, Mohamed Najib" sort="Marzouki, Mohamed Najib" uniqKey="Marzouki M" first="Mohamed Najib" last="Marzouki">Mohamed Najib Marzouki</name></author></analytic><series><title level="j">Biotechnology and applied biochemistry</title><idno type="eISSN">1470-8744</idno></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antibodies (genetics)</term><term>Antibodies (isolation & purification)</term><term>Antibodies (metabolism)</term><term>Gene Expression</term><term>Pichia (genetics)</term><term>Pichia (metabolism)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Scorpions (genetics)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antibodies</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Antibodies</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Antibodies</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Pichia</term><term>Scorpions</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Pichia</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Gene Expression</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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