N‐terminal chemical protein labeling using the naturally split GOS‐TerL intein

Abstract

Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

“…The ability of inteins to seamlessly ligate two peptides or to selectively release the N-or C-extein portion has been extensively exploited for protein modification and purification, to produce intein-based biosensor and reporter systems, and even in gene therapy [6][7][8][9][10][11][12][13] .…”

A mesophilic cysteine-less split intein for protein trans -splicing applications under oxidizing conditions

“…The required in vitro addition of reducing agents like dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine (TCEP), however, has detrimental effects on intein-fused POIs that are sensitive to reduction, for example by breaking structurally critical disulfide bonds, such as in antibodies ( Fig. 1A) (15)(16)(17). Moreover, such cysteine-dependent inteins will at best be only partially active in oxidizing environments like the endoplasmic reticulum, bacterial periplasmic space, or the in vivo extracellular milieu (18)(19)(20).…”

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

“…[100] While EPL facilitates the C-terminal functionalization of proteins,B achmann et al recently made use of the GOS-TerL intein and ap rotein trans-splicing reaction to functionalize aG FP-binding nanobody with as ynthetic fluorophore at its Nterminus. [101] However,w hen choosing EPL or intein-fusion strategies,r efolding from inclusion bodies is often required to achieve functional proteins, which significantly increases experimental effort. [102] In recent years,h uge efforts have been made to broaden the toolbox of conjugation methods for the functionalization of nanobodies and other antigen-binding proteins.W hile all of the methods discussed in this review have their pros and cons,t he methodological versatility enables the interested scientist to pick one of the validated techniques according to the requirements of the individual application.…”