‘Traceless’ tracing of proteins – high-affinity trans-splicing directed by a minimal interaction pair

Braner, Markus; Kollmannsperger, Alina; Wieneke, Ralph; Tampé, Robert

doi:10.1039/c5sc02936h

Cited by 30 publications

(32 citation statements)

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“…In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 36 , ion channels [37][38][39] , and G protein-coupled receptors 40,41 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]42 or by adding affinity tags to promote split intein interactions 18 . Such improvements would allow the approach to be applied to a broader complement of target proteins.…”

Section: Discussionmentioning

confidence: 99%

“…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 .…”

Section: Introductionmentioning

confidence: 99%

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

Khoo

Galleano

Gasparri

et al. 2020

Preprint

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AbstractManipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Khoo

Galleano

Gasparri

et al. 2020

Preprint

Self Cite

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“…Aside from the studies presented here,t he value of the trisNTA-His-tag interaction is further revealed by its application in 1) protein tethering to membranes, [12b,64] liposomes, [6b] glass surfaces, [65] gold interfaces, [9a, 66] and nanoparticles, [67] 2) guided high-affinity protein trans-splicing and "traceless" labeling, [56] 3) stochastic sensing of single mole- Figure 5. Fori nstance,t he genetic modification of target cells,o ften the need for cell fixation before imaging,p ossible inaccessibility of the His-tag due to steric shielding or unexpected changes of protein properties (for example,d ecreased solubility,m isfolding,o rd imerization) need to be considered before implementation of this method.…”

Section: Discussionmentioning

confidence: 94%

Multivalent Chelators for In Vivo Protein Labeling

Wieneke

Tampé

2019

Angew Chem Int Ed

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With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.

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“…The MCHs contain two to six NTA units, leading to (sub‐)nanomolar binding affinity towards a histidine tag for site‐specific labeling of targets even in a crowded cellular environment with low toxicity . The combination of high‐affinity binding properties and an ultra‐small, non‐disturbing affinity tag of smaller than 1 kDa enables 1) protein labeling with organic dyes, quantum dots, and streptavidin, 2) protein tethering to membranes, liposomes, glass surfaces, gold interfaces, and nanoparticles, 3) guided high‐affinity protein trans‐splicing and “traceless” labeling, and 4) stochastic sensing of single molecules in nanopores, single‐molecule tracking, or super‐resolution microscopy even in living cells . However, the variety of arrangements for connecting the NTA groups has led to a confusing array of MCH probes on offer, sometimes even sharing the same designation, despite a fundamentally different chemical structure.…”

Section: Figurementioning

confidence: 99%

The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His‐tagged Proteins in vitro and in Cells

et al. 2018

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Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal‐to‐background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N‐nitrilotriacetic acid (NTA)‐based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His‐tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low‐molecular‐weight interaction pairs.

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‘Traceless’ tracing of proteins – high-affinity trans-splicing directed by a minimal interaction pair

Abstract: Using a minimal lock-and-key element the affinity between the intein fragments for N-terminal protein trans-splicing was significantly increased, allowing for site-specific, ‘traceless’ covalent protein labeling in living mammalian cells at nanomolar probe concentrations.

Cited by 30 publications

References 50 publications

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

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

Multivalent Chelators for In Vivo Protein Labeling

The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His‐tagged Proteins in vitro and in Cells

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