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Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector

Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector

Authors :
Matthijs Wesseling
Peter Kronenberger
Philippe Giron
Fatima Haddouchi
Gudrun Walser
Albert-Menno Laffra
Alfiah Noor
Jacques De Greve
Source :
BMC Research Notes, Vol 11, Iss 1, Pp 1-7 (2018), BMC Research Notes
Publication Year :
2018
Publisher :
Springer Science and Business Media LLC, 2018.

Abstract

Objective Our aim was to produce a mono-biotinylated single domain antibody (‘nanobody’) specific for the epidermal growth factor receptor (EGFR), which is overexpressed in many cancer cells. The binding of the nanobody and its function are tested in cancer cells. The construct could be used to carry variable therapeutic or diagnostic load using biotin-streptavidin bridging. Results The EGFR-specific 7D12 nanobody was genetically fused to an IgA hinge linker and to a C-terminal biotin ligase acceptor sequence, allowing mono-biotinylation in E. coli. Expression was in strain BL21-DE3 from a T7 RNA polymerase driven pET22b vector. The biotinylated nanobody, isolated from the periplasm, was purified using streptavidin-mutein affinity chromatography. Final yields were up to 5 mg/l of cell culture. We showed that the construct could bind to EGFR expressing A431 epidermoid carcinoma cells, and to transiently transformed EGFR overexpressing HEK293T cells and not to EGFR negative control cells. The specificity for the EGFR was further demonstrated by immunoprecipitation. To test the functionality, PC9 non-small cell lung cancer cells were treated with mono-biotinylated nanobody or with streptavidin-coupled tetravalent nanobodies. Both were able to block mutant EGFR phosphorylation and slow down growth of PC9 cells. Tetravalent nanobodies were able to downregulate AKT phosphorylation. Electronic supplementary material The online version of this article (10.1186/s13104-018-3852-1) contains supplementary material, which is available to authorized users.

Details

ISSN :
17560500
Volume :
11
Database :
OpenAIRE
Journal :
BMC Research Notes
Accession number :
edsair.doi.dedup.....a28cc7b45e1d96a6f1b0ce6e8709c919
Full Text :
https://doi.org/10.1186/s13104-018-3852-1