Generation of an alpaca‐derived nanobody recognizing γ‐H2AX

Autor: Heinrich Leonhardt, Florian D. Hastert, Katrin Schmidthals, M. Cristina Cardoso, Oliver Mortusewicz, Malini Rajan, Ulrich Rothbauer, Alexander Rapp
Rok vydání: 2015
Předmět:
XRCC1
X-ray repair cross-complementing protein 1
Laser microirradiation
QH301-705.5
DNA repair
cells
Live cell microscopy
Method
HEK293
human embryonic kidney 293 cells
environment and public health
General Biochemistry
Genetics and Molecular Biology
Epitope
RFP
red fluorescent protein
chemistry.chemical_compound
Antigen
CKM
casein kinase 2 mutant
ELISA
enzyme linked immunosorbent assay
Biology (General)
siRNA
short interfering RNA
GFP
green fluorescent protein
MDC1
mediator of DNA damage checkpoint-1
biology
Alpaca heavy chain antibodies
HEK 293 cells
FRAP
fluorescence recovery after photobleaching
Transfection
VHH
variable domain of heavy-chain antibody
MEF
mouse embryonic fibroblast
Molecular biology
In vitro
H2AX
histone H2AX
enzymes and coenzymes (carbohydrates)
chemistry
biology.protein
KLH
keyhole limpet hemocyanin
biological phenomena
cell phenomena
and immunity
Antibody
Chromobodies
DNA
Post-translational modifications
Zdroj: FEBS Open Bio
FEBS Open Bio, Vol 5, Iss 1, Pp 779-788 (2015)
ISSN: 2211-5463
Popis: Highlights • An alpaca-derived γ-H2AX nanobody was generated. • γ-H2AX chromobody was able to bind and precipitate phosphorylated H2AX peptide. • γ-H2AX chromobody could be produced in bacterial as well as mammalian cells. • Alternative epitope recognition by γ-H2AX chromobody was induced by ectopic XRCC1. • Accessibility of γ-H2AX chromobody was hindered by MDC1 masking in vivo.
Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.
Databáze: OpenAIRE
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