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Directed organisation of protein complexes

Controlled protein immobilisation is an essential technique for many biotechnological applications. A European project attempted to develop a system to control the structural assembly of a multiple protein complex.
Directed organisation of protein complexes
Defined spatial organisation is the key in modern biotechnological applications of proteins, including the immobilisation of antibodies or creation of nanostructures on bio-templates. Most current techniques do not allow controlled co-immobilisation at the molecular scale of several proteins.

The EU-funded DECODEB (Defined co-immobilization by DNA binding protein tags) project developed a technology to create more advanced quaternary structures of proteins both in solution and on surfaces. The main objective was to create a protein fusion tag system allowing the co-immobilisation of proteins through protein-DNA interactions. DNA binding proteins recognising different sequences with high binding affinities were used as fusion tags to provide the sequence specific protein-DNA interaction link.

Variants of three proteins tags were synthesised, expressed and purified and their binding was assessed with electrophoretic mobility shift assay. All the variants of the DNA-binding tags demonstrated highly specific simultaneous co-binding to various double stranded DNA probes containing their DNA sequences.

For proof of concept, researchers tested fusion strategy with mediated protein ligation and controlled chemical crosslinking of native target proteins with DNA binding protein tags. This approach proved to be the most successful and was adopted by the project.

The designed co-immobilisation system was successfully employed for signal amplification in DNA biosensor development. The successful use of the peroxidase-reporter conjugate for signal amplification in the papilloma virus HPV16 genosensor opened the possibility for its application as a universal detection molecule. In conclusion, DECODEB activities demonstrated the utility of the conjugates for target molecule detection and signal amplification.

Related information

Keywords

Protein complexes, protein immobilisation, DECODEB, DNA-binding tag, DNA biosensor
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