Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code

Abstract

Conditional control of protein function in a living model organism is an important tool for studying the effects of that protein during development and disease. In this chapter, we walk through the steps to generate a small-molecule-activatable enzyme in zebrafish embryos through the incorporation of a noncanonical amino acid into the protein active site. This method can be applied to many enzyme classes, which we highlight with temporal control of a luciferase and a protease. We demonstrate that strategic placement of the noncanonical amino acid completely blocks enzyme activity, which is then promptly restored after addition of the nontoxic small molecule inducer to the embryo water.

Keywords: Conditional control; Genetic code expansion; Non-canonical amino acid; Staudinger reduction; Zebrafish.