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bioRxiv · 10.1101/2021.11.04.467337

A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli

Wu, T.·Joseph, S.
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Abstract

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Foster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but its technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

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Wu, T., Joseph, S.. 2021-11-04. A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli. https://doi.org/10.1101/2021.11.04.467337

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