Switchable inteins for conditional protein splicing

Ventura, Barbara Di; Mootz, Henning D.

doi:10.1515/hsz-2018-0309

Cited by 41 publications

(22 citation statements)

References 58 publications

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“…This could be achieved by means of light-sensitive protein domains that regulate intein splicing (39,40) or small molecule ligands that act directly on the split inteins (41) to control the protein-protein association step that precedes protein trans -splicing. Fast, accurate switching on of enzyme activity in vivo could be achieved if methods for efficient conditional activation of protein trans -splicing were available (42). An engineered photo-activatable gp41-1 split intein system was recently shown to work in bacterial cells (43), demonstrating the feasibility of this approach.…”

Section: Resultsmentioning

confidence: 99%

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Olorunniji

Lawson-Williams

McPherson

et al. 2019

Nucleic Acids Research

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Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

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Section: Resultsmentioning

confidence: 99%

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Olorunniji

Lawson-Williams

McPherson

et al. 2019

Nucleic Acids Research

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“…A proof-of-concept attempt to engineer switchable inteins by transposon-mediated domain-insertions. 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.…”

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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“…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 ( Supplementary Fig.…”

Section: Mitigation Of Undesirable Basal Activities In Highly Active mentioning

confidence: 99%

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Tyh

Shao

et al. 2020

Preprint

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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 augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

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Switchable inteins for conditional protein splicing

Cited by 41 publications

References 58 publications

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

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

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

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