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

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.

RNA Cloaking by Reversible Acylation

“…Staudinger reduction of the azide could result in cyclization, releasing restored RNA and lactam 2 (Figure 1b-c). Azide reduction with phosphines has been studied for multiple biological and biotechnological applications: for example, for reduction of azidophenylalanine in proteins, [19] and as a reversible terminator in sequencing chemistry. [7] O -azidomethylbenzoyl groups were previously used in organic synthesis to protect alcohols and amines, and were released during the deprotection step with aryl phosphines.…”

Efficient and wash‐free labeling of membrane proteins using engineered Npu DnaE split‐inteins

“…The use of short synthetic peptides opens multiple possibilities, by allowing the introduction of synthetic tags of various physicochemical properties to cellular proteins . However, this approach is mainly suitable for labeling of type II membrane proteins and inappropriate for labeling of type I membrane proteins . For general applications of this strategy in the monitoring of membrane proteins, the use of shorter N‐intein fragments also needs to be considered.…”

“…Although this approach was especially useful for N‐terminal labeling of type I membrane proteins, it required a considerable amount of time for labeling. Alternatively, Mootz's group demonstrated an efficient labeling approach utilizing click or amine tags introduced to a recombinant N‐intein . These intein‐mediated approaches that introduce chemical probes to target proteins in vivo offer a useful tool for various applications, but there are still some complications to overcome.…”

“…Alternatively, Mootz's group demonstrated an efficient labeling approach utilizing click or amine tags introduced to a recombinant N-intein. 25 These intein-mediated approaches that introduce chemical probes to target proteins in vivo offer a useful tool for various applications, but there are still some complications to overcome. In this work, we aimed to design a fast and efficient labeling method using engineered splitinteins to make this approach more applicable.…”

Drug Resistance in Bacteria, Fungi, Malaria, and Cancer

“…Synthetic biology approaches have provided efficient means for such manipulations, for example, incorporation of nonnatural amino acids and chemicals to produce synthetic proteins that do not occur in nature and have better potential to carry out their functions (Lang and Chin 2014;Schutz and Mootz 2014;Lang et al 2015). Another utilization of synthetic proteins is in production of chemicals and drugs.…”