Controllable intein splicing and N-terminal cleavage at mesophilic temperatures
Inteins (intervening proteins) interrupt host proteins and are removed through a protein splicing reaction that ligates adjacent N- and C-exteins. The ability of inteins to specifically rearrange peptide bonds has proven exceptionally useful in protein engineering, thus, methods to control intein ac...
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| Format: | Article |
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Frontiers Media S.A.
2025-02-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1543573/full |
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| author | Taylor A. McNeal Joel Weinberger Geraldy L. S. Liman Tia M. Ariagno David W. Wood Thomas J. Santangelo Christopher W. Lennon |
| author_facet | Taylor A. McNeal Joel Weinberger Geraldy L. S. Liman Tia M. Ariagno David W. Wood Thomas J. Santangelo Christopher W. Lennon |
| author_sort | Taylor A. McNeal |
| collection | DOAJ |
| description | Inteins (intervening proteins) interrupt host proteins and are removed through a protein splicing reaction that ligates adjacent N- and C-exteins. The ability of inteins to specifically rearrange peptide bonds has proven exceptionally useful in protein engineering, thus, methods to control intein activity are of considerable interest. One particularly useful application of inteins is for the removal of an affinity tag following purification of a target protein through N-terminal cleavage (NTC). Typically, extended incubation at high temperature (greater than 50°C) or with an external nucleophile (e.g., dithiothreitol) is required to drive NTC, conditions that compromise the folding of many target proteins. Here, we characterize a variant of the Thermococcus kodakarensis RadA intein that can perform NTC at moderate temperatures in the absence of an external nucleophile. While we find that while NTC is largely inhibited during expression in Escherichia coli at 15°C, rapid and efficient NTC can be activated 37°C. Our results provide an alternative intein-based system – one that does not require either an external nucleophile or prolonged incubation at high temperature to stimulate NTC – that controls intein activity within a temperature range amenable to most mesophilic experimental organisms. |
| format | Article |
| id | doaj-art-ed7f61c536bc4b9599ef1549798bbfab |
| institution | Kabale University |
| issn | 2296-4185 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-ed7f61c536bc4b9599ef1549798bbfab2025-02-07T06:49:38ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852025-02-011310.3389/fbioe.2025.15435731543573Controllable intein splicing and N-terminal cleavage at mesophilic temperaturesTaylor A. McNeal0Joel Weinberger1Geraldy L. S. Liman2Tia M. Ariagno3David W. Wood4Thomas J. Santangelo5Christopher W. Lennon6Department of Biological Sciences, Murray State University, Murray, KY, United StatesDepartment of Biological Sciences, Murray State University, Murray, KY, United StatesDepartment of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, United StatesDepartment of Biological Sciences, Murray State University, Murray, KY, United StatesWilliam G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, United StatesDepartment of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, United StatesDepartment of Biological Sciences, Murray State University, Murray, KY, United StatesInteins (intervening proteins) interrupt host proteins and are removed through a protein splicing reaction that ligates adjacent N- and C-exteins. The ability of inteins to specifically rearrange peptide bonds has proven exceptionally useful in protein engineering, thus, methods to control intein activity are of considerable interest. One particularly useful application of inteins is for the removal of an affinity tag following purification of a target protein through N-terminal cleavage (NTC). Typically, extended incubation at high temperature (greater than 50°C) or with an external nucleophile (e.g., dithiothreitol) is required to drive NTC, conditions that compromise the folding of many target proteins. Here, we characterize a variant of the Thermococcus kodakarensis RadA intein that can perform NTC at moderate temperatures in the absence of an external nucleophile. While we find that while NTC is largely inhibited during expression in Escherichia coli at 15°C, rapid and efficient NTC can be activated 37°C. Our results provide an alternative intein-based system – one that does not require either an external nucleophile or prolonged incubation at high temperature to stimulate NTC – that controls intein activity within a temperature range amenable to most mesophilic experimental organisms.https://www.frontiersin.org/articles/10.3389/fbioe.2025.1543573/fullinteinprotein splicingN-terminal cleavagebioseparationsbiosensor |
| spellingShingle | Taylor A. McNeal Joel Weinberger Geraldy L. S. Liman Tia M. Ariagno David W. Wood Thomas J. Santangelo Christopher W. Lennon Controllable intein splicing and N-terminal cleavage at mesophilic temperatures Frontiers in Bioengineering and Biotechnology intein protein splicing N-terminal cleavage bioseparations biosensor |
| title | Controllable intein splicing and N-terminal cleavage at mesophilic temperatures |
| title_full | Controllable intein splicing and N-terminal cleavage at mesophilic temperatures |
| title_fullStr | Controllable intein splicing and N-terminal cleavage at mesophilic temperatures |
| title_full_unstemmed | Controllable intein splicing and N-terminal cleavage at mesophilic temperatures |
| title_short | Controllable intein splicing and N-terminal cleavage at mesophilic temperatures |
| title_sort | controllable intein splicing and n terminal cleavage at mesophilic temperatures |
| topic | intein protein splicing N-terminal cleavage bioseparations biosensor |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1543573/full |
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