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bioRxiv · 10.1101/2022.05.19.492583

Creating a thermostable beta-glucuronidase switch for homogeneous immunoassay by disruption of conserved salt bridges at diagonal interfaces

Zhu, B.·Cheng, Q.·Tang, H.·Kitaguchi, T.·Ueda, H.
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Abstract

The Escherichia coli beta-glucuronidase (GUS) has been used as a reporter enzyme in molecular biology and has been engineered to enzyme switches for the development of homogeneous biosensors. Here, we developed a thermostable GUS enzyme switch from a thermostable GUS mutant TR3337 by disrupting a conserved salt bridge (H514-E523) between the diagonal subunits of its homotetramer. A combinatorial library (240 variants) was screened by a novel high-throughput strategy, and a mutant DLW (H514D/M516L/Y517W) was found to be a functional enzyme switch in a caffeine-recognizing immunosensor. The molecular dynamics simulations were performed to predict the topology change around position 514, and the sidechain flip of D514 (repulsion with E523) was observed in the DLW mutant. Up to 1.8-fold of the signal-to-background ratio was confirmed when measured at 45 {degrees}C, which makes the DLW mutant a versatile tool for developing the thermostable immunosensors for in vitro and in cellulo applications. Table of contents graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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Zhu, B., Cheng, Q., Tang, H., Kitaguchi, T., Ueda, H.. 2022-05-20. Creating a thermostable beta-glucuronidase switch for homogeneous immunoassay by disruption of conserved salt bridges at diagonal interfaces. https://doi.org/10.1101/2022.05.19.492583

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