Utilizing intein trans-splicing for in vivo generation of site-specifically modified proteins

Maksimovic, Igor; Ray, Devin M.; Zheng, Qingfei; David, Yael

doi:10.1016/bs.mie.2019.07.015

Cited by 3 publications

(3 citation statements)

References 20 publications

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“…A key aspect of intein chemistry allows for ChemBioChem the site specificity in the introduction of the desired modification. [24] While in this method we utilized split-inteins to modify a target protein, the system enables labeling of a precise residue of the desired protein when designed correctly. As split inteins continue to be adapted to cellular systems, the next big advancement will be taking advantage of their site specificity, such as by incorporating a specific post-translational modifica-tion or photo-crosslinker to a protein domain.…”

Section: Discussionmentioning

confidence: 99%

“…Inteins represent an exciting space in the quest to engineer proteins in live cells. A key aspect of intein chemistry allows for the site specificity in the introduction of the desired modification [24] . While in this method we utilized split‐inteins to modify a target protein, the system enables labeling of a precise residue of the desired protein when designed correctly.…”

Section: Discussionmentioning

confidence: 99%

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Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

2022

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Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogon-ality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair Ava N /Npu C can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

2022

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“…There is a critical need for novel approaches to study NECMs including the development of high-resolution trace mass spectrometry, chemical probes for specific enrichment, and site-specific antibodies. Individual and customized NECMs can also be specifically introduced into designated targets in vivo using intein-mediated protein splicing (Maksimovic et al, 2019) and amber codon suppression (Zhang et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Non-enzymatic covalent modifications: a new link between metabolism and epigenetics

et al. 2020

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Epigenetic modifications, including those on DNA and histones, have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways. In turn, metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications. Recently, non-enzymatic covalent modifications (NECMs) by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms. Here, we summarize these recent advances in the identification and characterization of NECMs on nucleic acids, histones, and transcription factors, providing an additional mechanistic link between metabolism and epigenetics.

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Efficient splicing of the CPE intein derived from directed evolution of the <italic>Cryptococcus neoformans</italic> PRP8 intein

Zhan¹,

Shi²,

Jiang³

et al. 2023

ABBS

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Intein-mediated protein splicing has been widely used in protein engineering; however, the splicing efficiency and extein specificity usually limit its further application. Thus, there is a demand for more general inteins that can overcome these limitations. Here, we study the trans -splicing of CPE intein obtained from the directed evolution of Cne PRP8, which shows that its splicing rate is ~29- fold higher than that of the wild-type. When the +1 residue of C-extein is changed to cysteine, CPE also shows high splicing activity. Faster association and higher affinity may contribute to the high splicing rate compared with wild-type intein. These findings have important implications for the future engineering of inteins and provide clues for fundamental studies of protein structure and folding.

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Utilizing intein trans-splicing for in vivo generation of site-specifically modified proteins

Cited by 3 publications

References 20 publications

Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Non-enzymatic covalent modifications: a new link between metabolism and epigenetics

Efficient splicing of the CPE intein derived from directed evolution of the <italic>Cryptococcus neoformans</italic> PRP8 intein

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Utilizing intein trans-splicing for in vivo generation of site-specifically modified proteins

Cited by 3 publications

References 20 publications

Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Non-enzymatic covalent modifications: a new link between metabolism and epigenetics

Efficient splicing of the CPE intein derived from directed evolution of the &lt;italic&gt;Cryptococcus neoformans&lt;/italic&gt; PRP8 intein

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Efficient splicing of the CPE intein derived from directed evolution of the <italic>Cryptococcus neoformans</italic> PRP8 intein