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Enhanced performance of a plate-based TEV protease assay using linker-optimized, site-specifically biotinylated
Mitsuki Nakamura1, Yoshiki Miya1, Masafumi Sakono1
1Department of Applied Chemistry, Faculty of Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan.
Abstract:
We developed a plate-based luminescence assay for TEV protease using NanoLuc substrates site-specifically biotinylated through BirA co-expression in Escherichia coli. Substrates containing a TEV recognition sequence and AviTag, with or without a G4S linker, were immobilized on streptavidin-coated plates. Protease activity was quantified from the luminescence of NanoLuc released after cleavage. G4S insertion improved biotinylation efficiency and the luminescence response of immobilized substrates. Four-parameter logistic analysis showed lower EC20 and EC50 values and improved limits of detection and quantification. These findings demonstrate that linker insertion enhances substrate preparation and analytical sensitivity in this assay.
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