Creation of stable and strictly regulated enzyme switch for signal-on immunodetection of various small antigens

Recently, we reported a fusion-protein-based immunodetection system comprising the two domains of an antibody variable region as the detectors, each tethered to an interface mutant β-glucuronidase (GUSm) as the reporter, for detecting small molecules via dimerization of dimer activation. However, the poor stability of GUSm and background signal propagation possibly due to spontaneous proteolysis undermined its performance. To solve these problems, we attempted thermostabilization of GUSm by using a previously isolated thermostable mutant GUS_IV5 as a backbone. After screening several interface mutants, we selected one with M516K/Y517W mutation because it exhibited higher activity after dimerization than the wild-type GUS, while maintaining very low background activity. By using this improved immunosensor, we achieved a two-fold improvement in terms of sensitivity in the detection of 4-hydroxy-3-nitrophenyl acetyl. Moreover, by constructing a new biosensor tethered to a nanobody for caffeine as the detector, we could achieve noncompetitive signal-on detection of caffeine in a practically useful concentration range.