Improved protein splicing using embedded split inteins

Gramespacher, Josef A.; Stevens, Adam J.; Thompson, Robert E.; Muir, Tom W.

doi:10.1002/pro.3357

Cited by 18 publications

(17 citation statements)

References 23 publications

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“…More importantly, techniques suitable in in vivo applications remain one of the key challenges in studying histone PTMs. [97][98][99][100][101][102][103][104] Therefore, we anticipate that more chemical methods would be developed to constitute a versatile toolbox that can facilitate the mechanistic understanding of histone PTM crosstalk.…”

Section: Discussionmentioning

confidence: 99%

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

Peng

2021

Journal of Peptide Science

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The reversible and dynamic post‐translational modifications (PTMs) of histones in eukaryotic chromatin are intimately connected to cell development and gene function, and abnormal regulation of PTMs can result in cancer and neurodegenerative diseases. Specific combinations of these modifications are mediated by a series of chromatin proteins that write, erase, and read the “histone codes,” but mechanistic studies of the precise biochemical and structural relationships between different sets of modifications and their effects on chromatin function constitute a unique challenge to canonical biochemical approaches. In the past decade, the development and application of chemical methods for investigating histone PTM crosstalks has received considerable attention in the field of chemical biology. In this review, taking the functional crosstalk between H2B ubiquitylation at Lys120 (H2BK120ub) and H3 methylation at Lys79 (H3K79me) as a typical example, we survey recent developments of different chemical methods, in particular, protein synthetic chemistry and protein‐based chemical probes, for studying the mechanism of the functional crosstalks of histone PTMs.

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Section: Discussionmentioning

confidence: 99%

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

Peng

2021

Journal of Peptide Science

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“…S16] was delivered via electroporation to cells expressing the corresponding VidaL C construct (FLAG-H3 1-28 -Vid C -H3 34-135 -GFP). Note that the C-intein was embedded within the histone tail in order to improve expression (35). After a biotin IP, we observed the presence of both PTMs by Western blotting (Fig.…”

Section: Significancementioning

confidence: 96%

Live-cell protein engineering with an ultra-short split intein

Burton

Haugbro

Parisi

et al. 2020

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

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Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient proteintrans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.

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“…This capability has been harnessed to assemble partial, non-functional Ca V and Na V channel fragments into full-length, functional channels using a single pair of split inteins (Subramanyam et al 2013;Lueck et al 2016). Given the increasing number of identified and splicing-optimized split inteins (Stevens et al 2016(Stevens et al , 2017Gramespacher et al 2018;Bhagawati et al 2019;Burton et al 2020;Pinto et al 2020), it is now also possible to use two orthogonal pairs of split inteins to insert a synthetic peptide between recombinantly expressed N-and C-terminal protein fragments (Fig. 5A).…”

Section: Protein Trans-splicingmentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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Improved protein splicing using embedded split inteins

Cited by 18 publications

References 23 publications

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

Live-cell protein engineering with an ultra-short split intein

The current chemical biology tool box for studying ion channels

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