a b s t r a c tWe have studied the naturally split a subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of ð1:1 AE 0:2Þ Â 10 À2 s À1 at 37°C. This represents the highest rate reported for the protein trans-splicing reaction so far (t 1=2 of $ 60 s). Furthermore, the reaction was very robust and highyielding with respect to different extein sequences, temperatures from 6 to 37°C, and the presence of up to 6 M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.

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.

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.

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.

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