Abstract
Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.
Highlights
Protein splicing is a unique post-translational phenomenon where an internal peptide sequence known as the intein, removes itself from a non-functional precursor protein while concurrently ligating the flanking precursor ends with a peptide bond to restore the reassembled precursor gene’s function [1,2,3]
The efficacy of protein trans-splicing (PTS) activity was assayed in live cell imaging by co-expressing the C-terminal precursor comprised of a truncated InC fused to Venus (i.e. t#-InC-Venus) with the N-terminal precursor comprised of a tandem fusion of the plasma membrane (PM) localization peptide from Lyn kinase (Lyn) [39] the cyan fluorescent protein mutant Cerulean
In the absence of PTS activity, the PM would be labeled with Cerulean and mRFP fluorescence while the cytoplasm was labeled with Venus fluorescence (Fig 1B–1D and 1L–1N)
Summary
Protein splicing is a unique post-translational phenomenon where an internal peptide sequence known as the intein, removes itself from a non-functional precursor protein while concurrently ligating the flanking precursor ends (i.e. exteins) with a peptide bond to restore the reassembled precursor gene’s function [1,2,3]. In exploiting this phenomenon, the ability to control protein splicing can offer new approaches of regulating protein activity.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
