Click‐Tag and Amine‐Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split <i>Npu</i> DnaE Intein

Schütz, Vivien; Mootz, Henning D.

doi:10.1002/ange.201309396

Cited by 10 publications

(8 citation statements)

References 46 publications

(24 reference statements)

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“…S10A). The internal Trx domain was previously found to support correct localization of such model receptor constructs in the plasma membrane and increase protein splicing activity (19,21). However, upon treating these cells with the fusion protein eGFP-CL N -H 6 (14) at concentrations up to 5 μM, we could detect neither any specific eGFP signal on the transfected cells via confocal microscopy (SI Appendix, Fig.…”

Section: Chemical Modification Of Cell-surface Proteins With a Minimamentioning

confidence: 83%

“…However, these tags are of considerable size and thus potentially interfere with receptor function. Previous reports to overcome this limitation by exploiting virtually traceless protein trans-splicing of very short fluorophore-conjugated tags used split inteins that contained catalytic cysteines and hence required the presence of reducing agents (19,21,22,(56)(57)(58).…”

Section: Chemical Modification Of Cell-surface Proteins With a Minimamentioning

confidence: 99%

“…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). Collateral damage by the reducing agents will also occur on other proteins when performing the trans-splicing reaction on an extracellular domain of a cell surface protein in a whole cell context (21,22). Virtually all characterized inteins face this limitation, including the most widely used split Npu DnaE intein and its engineered variants (17,23,24), as well as the split Gp41-1 intein (25,26), the fastest splicing intein known to date.…”

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confidence: 99%

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

Bhagawati

Tme

Füsser

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody–epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

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Section: Chemical Modification Of Cell-surface Proteins With a Minimamentioning

confidence: 83%

Section: Chemical Modification Of Cell-surface Proteins With a Minimamentioning

confidence: 99%

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confidence: 99%

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

Bhagawati

Tme

Füsser

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…However, tPTS has largely been conducted in vitro or restricted to bacterial expression systems, cell lysates, nuclear extracts, or selection protocols 8,[13][14][15] . Indeed, most live-cell applications of PTS utilize single split inteins for the purpose of N/C-terminal tagging [16][17][18] or manipulating protein assembly/expression 19,20 .…”

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confidence: 99%

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

et al. 2020

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Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, Na V 1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

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“…Notably, cysteine residues in the protein of interest will not be affected. The CysTag approach was conceptually extended to the AmineTag and ClickTag approaches to circumvent the lack of suitable intein fragments devoid of cysteines . However, these latter two approaches are significantly more elaborate on the technical level.…”

Section: Introductionmentioning

confidence: 99%

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

Bachmann

Mootz

2017

Journal of Peptide Science

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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.

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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

Cited by 10 publications

References 46 publications

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

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

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

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

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