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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
- 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.
- Subjects :
- Single-domain antibody
0301 basic medicine
Immunoprecipitation
EGFR
lcsh:Medicine
General Biochemistry, Genetics and Molecular Biology
03 medical and health sciences
0302 clinical medicine
Mono-biotinylation
Cell Line, Tumor
Escherichia coli
Humans
Epidermal growth factor receptor
lcsh:Science (General)
lcsh:QH301-705.5
Cancer
VHH domain
biology
Chemistry
lcsh:R
HEK 293 cells
General Medicine
Single-Domain Antibodies
Molecular biology
ErbB Receptors
Research Note
HEK293 Cells
030104 developmental biology
lcsh:Biology (General)
Epidermoid carcinoma
Cell culture
030220 oncology & carcinogenesis
Biotinylation
Periplasm
Cancer cell
Nanobody
biology.protein
Streptavidin
Nanocarriers
lcsh:Q1-390
Subjects
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