Proximity Induced Splicing Utilizing Caged Split Inteins

Gramespacher, Josef A.; Burton, Antony J.; Guerra, Luis F.; Muir, Tom W.

doi:10.1021/jacs.9b05721

Cited by 18 publications

(23 citation statements)

References 36 publications

(55 reference statements)

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“…In theory, our IBM workflow should allow one to use a conditional intein [46][47][48] in place of a split intein, and then screen for insertion or split sites that would enable post-translational control of protein function. We thus synthesized and tested three reported chemogenic conditional inteins [49][50][51] from the literature, but they did not work well under our specific context in E. coli (Supplementary Fig. 18).…”

Section: Resultsmentioning

confidence: 99%

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Shao

et al. 2021

Nat Commun

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Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.

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Section: Resultsmentioning

confidence: 99%

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Shao

et al. 2021

Nat Commun

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“…To screen the GR effectors, we designed a sensing system based on the well-studied pair of Nostoc punctiforme (Npu) DnaE split inteins, which mediate fast and efficient protein trans-splicing (PTS) reactions [ 30 , 31 , 32 ]. The key to our approach is that cortisol-bound GR translocates to the nucleus, triggering CPS to reconstitute the split signal peptide to transport the fluorescent cargo ( Scheme 2 ).…”

Section: Resultsmentioning

confidence: 99%

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

Ryu

Lee

Kang

et al. 2021

IJMS

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Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

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“…Indeed, the small size of VidaL N will likely be advantageous in this regard. The technology described herein can also be extended through combination with conditionally splicing intein designs, enabling the triggering of PTS at selected time points or a chosen cellular state (44). Moreover, the combination of the approach described here with dCas9-guided localization could be exploited to enable the precise chemical modification of selected genomic regions or even single loci-an ongoing challenge in genome engineering (45).…”

Section: Discussionmentioning

confidence: 99%

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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Proximity Induced Splicing Utilizing Caged Split Inteins

Cited by 18 publications

References 36 publications

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

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

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