| Popis: |
Split enzymes have been used in Protein Complementation Assays (PCA) for several years to study protein-protein interactions. Recently Affimers, non-antibody binding proteins, have been created as new tools for studying molecular interactions. Combining these technologies offers substantial benefits for development of highly sensitive and specific diagnostic devices. For proof-of-principle, two fragments of β-lactamase have been generated with two His tags. The binding of the His tag on each fragment to nickel ions facilitates the association of β-lactamase fragments to generate a functional enzyme capable of substrate turnover. Substrates, such as the cephalosporin nitrocefin giving rise to a colour change (yellow to red) detectable at 492 nm. Following this proof of principle, the project focuses upon a novel split alkaline phosphatase to underpin an amperometric detection system developed with colleagues in the School of Electronic Engineering, University of Leeds. Alkaline phosphatase is a homodimer and the monomeric form of this protein does not turnover substrate. Current work in this study is focused on engineering the dimer interface to generate novel monomers that do not spontaneously associate. This is followed by exploring whether the mutated monomers can become associated and brought together, and catalyse substrate turnover. This study also focuses on preparing Affimers that can bind a target molecule, mGFP, at two separate epitopes. Followed by generating a cross linked protein between the Affimers and alkaline phosphatase. The binding of Affimer to target will be amplified by enzyme activity allowing detection. The system should allow single step detection of a target present at very low concentrations in a complex sample. |
