Chemical-tag labeling of proteins using fully recombinant split inteins.

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.

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.

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.

The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction.

We have studied the naturally split alpha 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 (1.1+/-0.2)x10(-2) s(-1) at 37 degrees C. This represents the highest rate reported for the protein trans-splicing reaction so far (t(1/2) of approximately 60s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37 degrees 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.