Evaluation of a novel affinity-label reporter protein with SNAP-tag and monomeric streptavidin

Reporter proteins are often used to detect pathogenic bacteria and viruses that are challenging to detect with straightforward assays. However, reporter protein assays are limited by their difficulty in detecting low levels of reporters in large sample sizes and/or in complex matrices. When reporter concentration techniques are employed, enzymatic reporters tend to lose their catalytic activity. In this study, we designed a heterobifunctional reporter composed of SNAP-tag and monomeric streptavidin (SNAP-mSA) that would allow its rapid concentration and easy detection. SNAP protein was used to capture the reporter using functionalized magnetic beads that offered an irreversible binding. Monomeric streptavidin was used to allow labeling the reporter instead of an enzymatic assay for detection. For the proof-of-principle, biotinylated horseradish peroxidase was used as the label for colorimetric detection. Immobilization of the reporter was achievable with a maximum loading of 1.5 μg/μL of magnetic beads for SNAP-mSA. The immobilized reporter was successfully labeled with biotinylated horseradish peroxidase and allowed for a detection limit of 0.2 ng SNAP-mSA. The reporter was able to retain the structure of their active sites after the washing steps and did not lose their binding ability to O6-benzylguanine (BG) and Biotin, respectively. SNAP-mSA was expressed through a T7 phage to determine its ability to detect E. coli BL21. The engineered phage could give a limit of detection of 105−6 CFU of E. coli BL21 in 7 h without an initial enrichment step. These findings suggest that SNAP-mSA can be used to successfully concentrate and label reporter proteins. •Reporter protein from SNAP-tag and monomeric streptavidin (mSA) can be expressed.•Expressed SNAP-mSA be immobilized on to SNAP-capture magnetic beads.•SNAP-mSA can be labeled with biotinylated horseradish peroxidase for detection.•T7 phage genome can be genetically engineered to express SNAP-mSA reporter protein.•Phage expressed SNAP-mSA allows a colorimetric detection of 105−6 CFU of E. coli.