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Cloning and expression of the ataxia-telangiectasia gene in baculovirus
S P Scott1, N Zhang, K K Khanna
1The Queensland Cancer Fund Research Laboratories, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, 4029, Australia.
Abstract:
The gene mutated in the human genetic disorder ataxia-telangiectasia, ATM, is implicated in the response to radiation-induced DNA damage and to a more widespread signalling defect. The ATM protein is predominantly a nuclear protein where it interacts with p53 and c-Abl as part of a radiation signal transduction pathway(s). We describe here the cloning of full-length ATM cDNA in a baculovirus vector to produce recombinant protein. Expression of ATM, as a soluble protein, was observed by 36 h post-infection using immunoblotting with anti-ATM antibody. The presence of a hexahistidine tag on ATM was used as the basis for purification of the protein by affinity chromatography. The protein yield was only 20 ng/100 ml of infected cells, presumably because of the size of the protein and adverse effects on cell growth when overexpressed. ATM was found to have autophosphorylation activity in immunoprecipitates with antibodies directed against the hexahistidine tag sequence. These results demonstrate that ATM can be expressed inefficiently in baculovirus infected insect cells and the data suggest that it phosphorylates itself.
Insights
Researchers cloned the ATM gene to study its role in DNA damage response. They successfully produced and purified the ATM protein, finding it possesses autophosphorylation activity, suggesting self-phosphorylation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Signaling
Background:
- The ATM gene is crucial for repairing radiation-induced DNA damage and is linked to ataxia-telangiectasia.
- ATM protein functions in nuclear signaling pathways, interacting with p53 and c-Abl during DNA damage response.
Purpose of the Study:
- To clone the full-length ATM cDNA and produce recombinant ATM protein using a baculovirus expression system.
- To characterize the biochemical properties of the recombinant ATM protein, specifically its autophosphorylation activity.
Main Methods:
- Full-length ATM cDNA was cloned into a baculovirus vector for expression in insect cells.
- Recombinant ATM protein was purified using affinity chromatography based on a hexahistidine tag.
- Immunoblotting and immunoprecipitation assays were used to detect and analyze ATM protein activity.
Main Results:
- Soluble recombinant ATM protein was successfully expressed and detected within 36 hours post-infection.
- Purified ATM protein exhibited autophosphorylation activity in vitro.
- Low protein yield was observed, attributed to ATM's large size and potential negative effects on host cell growth.
Conclusions:
- ATM can be expressed, albeit inefficiently, in baculovirus-infected insect cells.
- The findings suggest that ATM protein possesses intrinsic autophosphorylation capabilities, playing a role in its own regulation.