A bio-orthogonal and covalent 5 kDa small protein tag
Pabst, U.; Rossius, J.; Olesen, C. H.; Birol, M.; Broichhagen, J.
Show abstract
Precise and minimally perturbative protein labelling remains a key challenge for studying biomolecular function in living systems. Here, a minimal way to specifically label proteins based on SNAP-tag and intein-mediated protein splicing reaction is introduced. Termed CLUSTER (for Chemical Label-Unfold-Splice Technology Enables Recombination), this chimeric platform supports efficient labelling across diverse targets in living cells by retaining a fluorescent, 5 kDa sized peptide on a fusion protein of interest after splicing. A bacterial screening workflow was developed to optimize the reaction efficiency and construct design. Quantitative characterization using fluorescence polarization provides mechanistic insight into labelling efficiency and dynamics, while molecular dynamics simulations elucidate its stability, grasping the intricate nature of protein behaviour upon covalent labelling. This bio-orthogonal labelling technology allows for a versatile and minimally invasive approach for protein labelling, providing a powerful tool to probe protein behavior in native cellular systems.
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