Click-Tag and Amine-Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein

摘要

Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans-splicing. For the amine~tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine-reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click-tag, protein trans-splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p-azidophenylalanine. By the two-step strategy, exposure of the protein of interest to the copper catalyst was avoided.