Development of a cell-permeable Biotin-HaloTag ligand to explore functional differences between protein variants across cellular generations

HaloTag technology represents a versatile tool for studying proteins. Fluorescent HaloTag ligands employed in sequential labeling led to the discovery of distinct protein variants for histones, cohesins, and MCM complexes. Nonetheless, an efficient biochemical approach to separate the distinct protein variants to study their biological functions is missing. Principally being a gap in technology, the HaloTag toolbox lacks affinity ligands displaying good cell permeability and efficient affinity capture. Here, we describe the design, synthesis, and validation of a new cell-permeable Biotin-HaloTag ligand, which allows rapid labeling of Halo-tagged proteins in live cells and their efficient separation using streptavidin pull-down. Our work outlines how to use the herein-developed affinity ligand in sequential labeling to biochemically separate distinct protein variants and study their biological properties. The approach holds immense potential for addressing fundamental questions concerning essential cellular processes, including genome duplication and chromatin maintenance.