Quantitative Analysis of Protein Bioconjugation in Cells with an Expanded Genetic Code

Abstract

Protein bioconjugation – specific covalent bond formation between a protein of interest (POI) and a label – has a major impact on protein imaging, protein function studies, and drug discovery. Ideal protein bioconjugation should be fast, quantitative, one step with no off‐target binding, and have minimal impact on protein function, no matter the complexity of the biological environment. The impressive selectivity and kinetics of bioorthogonal reactions, specifically the catalyst‐free inverse‐electron demand Diels‐Alder (IEDDA) cycloaddition of tetrazines and strained alkene dienophiles, make IEDDA‐reactive unnatural amino acids an exceptional tool for protein bioconjugation via genetic code expansion. Analyses of these bioconjugation reactions in live cells has been mostly limited to fluorophore labeling, and thus is rarely quantitative. Self‐labeling enzymes, like HaloTag, have been used for protein isolation and purification, studies of protein‐protein interactions, and in vivo and in vitro imaging. The excellent permeability and low reactivity of the chloroalkane substrate of HaloTag yields bioconjugation detection independent of ligand permeability. The objective of this research is to utilize the HaloTag technology in combination with genetic code expansion to develop a generalized method for quantification of intracellular bioconjugation to any POI at defined sites. We hypothesize that bioconjugation efficiency could be assessed through site‐specific installation of a reactive handle on a POI, followed by ligand‐mediated protein labeling via ternary complex formation after cell lysis and exposure to HaloTag. Here we describe the optimization of concentrations, reaction times, and reaction partners for bioconjugation and pulldown for POIs expressing different IEDDA reactive surface residues. Additionally, we describe the attempted application of this method for labeling protein targets, such as kinases and nuclear receptors. With this new methodology, we have demonstrated quantitative assessment of protein bioconjugation reactions in live cells.