DECODEB · Defined co-immobilization by DNA binding protein tags

Defined co-immobilization by DNA binding protein tags

Defined spatial distribution of proteins plays a major role in nature, like in structures responsible for bacterial cell mobility, capsid structures of viruses, structures responsible for the controlled segregation of chromosomal DNA both in eukaryotic and prokaryotic cells and in membrane protein complexes required for protein and nutrient transport. Protein immobilization is also a key technique for many biotechnological applications, for example in protein microarrays and electrode surfaces for biosensor development, and more recently in engineered metabolic pathways for increased production of metabolites and the creation of nanostructures through biotemplating. A wide variety of strategies have been developed to immobilize proteins to surfaces, through physical adsorption, covalent crosslinking and bioaffinity interactions. In general though, these techniques are suitable for the immobilization of a single protein and do not allow the controlled co-immobilization at the molecular scale of several proteins, whereas the full retention of protein conformation and activity cannot be assured. The DECODEB project aspires to develop a system that goes beyond single protein immobilization and allows to control, in a simple and straightforward way, the structure of a multiple protein complex and to create more advanced quaternary structures at the protein level both in solution and on surfaces. The overall objective is to develop a protein fusion tag system that will allow the defined co-immobilization of proteins by means of protein-DNA interactions. A collection of DNA binding proteins with different sequence specificities and high binding affinities will be developed and used as fusion tags to target proteins, ultimately providing the sequence specific protein-DNA interaction link. According to the work plan, the specific goals of the project are: - Selection and development of high affinity DNA binding protein tags - Preliminary characterization of the co-immobilization system - Practical application of the system - Exploration of further applications Initially, a panel of DNA binding proteins that fulfilled several criteria regarding DNA binding sequence specificity and affinity, and good folding properties facilitating their heterologous soluble expression in Escherichia coli and downstream purification was selected: (i) a dimeric single chain variant of the bacteriophage lambda Cro repressor protein (scCro16), (ii) the monomeric stage III sporulation protein D from Bacillus subtilis (SpoIIID) and (iii) the DNA binding domain of the lac repressor protein (LacI) from Escherichia coli. Several variants of the three proteins tags were engineered, expressed and purified and their binding properties were assessed with electrophoretic mobility shift assays (EMSA). All variants of the chosen DNA binding protein tags were able to simultaneously co-bind to various dsDNA probes containing their specific DNA binding sequences, whereas non-specific binding to irrelevant DNA sequences was not observed. The proof-of-concept for defined protein co-immobilization was achieved with a bi-enzymatic metabolic pathway used by Escherichia coli to synthesize trehalose from glucose 6-phosphate and uridine diphosphate glucose. The two enzymes participating in this pathway trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP) were expressed in Escherichia coli as genetic fusions with the DNA binding protein tags scCro16 and SpoIIID. EMSA assays demonstrated that the enzyme fusions could be successfully co-immobilized on dsDNA probes containing the specific binding sites for the corresponding DNA binding protein tags, but enzyme activity assays revealed that this co-immobilization did not result in increased trehalose production. Further investigation indicated that the decreased enzyme activity was probably due to conformational changes after genetically fusing the enzymes to the DNA binding protein tags. In order to bypass genetic fusion of the protein targets with the DNA binding protein tags, alternative fusion strategies were explored, like sortase-A mediated protein ligation and controlled chemical crosslinking of native target proteins with specifically engineered DNA binding protein tags. The latter approach proved to be the most successful and was therefore adapted. The proposed co-immobilization system was also used successfully for signal amplification of a DNA biosensor. Horseradish peroxidase (HRP), one of the most common reporter enzymes used in biosensors, was conjugated to engineered DNA binding protein tag scCro16 in order to obtain a conjugate at a 1:1 stoichiometry. The target molecule was high-risk Human Papillomavirus type 16 ssDNA that was detected with a colorimetric Enzyme Linked Oligonucleotide Assay (ELONA). Specific capture and detection probes were designed based on the DNA sequence of the E7 oncogene protein of HPV16. The ssDNA detection probe was used as a direct HRP conjugate and as a ssDNA-dsDNA hybrid with the dsDNA extension containing multiple binding sites for scCro16. EMSA assays verified the specific binding of the HRP-scCro16 conjugate to dsDNA probes containing the scCro16 binding sequence whereas non-specific binding was not observed. Using the hybrid ssDNA-dsDNA detection probe with multiple binding sites for scCro16 and the HRP-scCro16 conjugate, it was possible to significantly improve the sensitivity of the genosensor and this improvement was directly related to the number of DNA binding sites included in the detection probe. Ultimately, the signal enhancement derived from the association of each ssDNA target with multiple conjugate molecules because of their simultaneous co-immobilization on the detection probe. The successful use of the HRP-scCro16 conjugate for signal amplification in the HPV16 genosensor has paved the way for its use as a universal detection molecule for various applications. The impact of DECODEB is based on the use of this conjugate or analogous ones (other reporter enzyme conjugated to a DNA binding protein tag with different sequence specificity) for target molecule detection and signal amplification, like other DNA targets or even protein targets using aptamers as detection molecules, bypassing the time-consuming and expensive preparation of direct reporter enzyme conjugates and functionalized nanoparticles.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз