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 |
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| Authors: | Dr Vivien Schütz Prof?Dr Henning D Mootz |
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| Affiliation: | Institute of Biochemistry, University of Muenster, Wilhelm‐Klemm‐Strasse 2, 48149 Münster (Germany) |
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| Abstract: | 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. |
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| Keywords: | bioconjugation bioorthogonality fusion tags lysine modification unnatural amino acids |
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