Sigma Xi Poster Competition
Controllable Intein Splicing and N-terminal Cleavage at Mesophilic Temperatures
Academic Level at Time of Presentation
Faculty/Staff
Major
Biomedical Sciences
Minor
Chemistry
List all Project Mentors & Advisor(s)
Dr. Christopher Lennon
Presentation Format
Poster Presentation
Abstract/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. Here, we characterize a variant of the Thermococcus kodakarensis RadA intein with the homing endonuclease (HEN) domain deleted that can perform controllable protein splicing and N-terminal cleavage (NTC) in the mesophilic temperature range. We find that intein activity is largely inhibited during expression in Escherichia coli at low temperature (15C) and activated upon a slight temperature increase (>20C). We observe a differential response to the external nucleophiles hydroxylamine and dithiothreitol, which suggests that the scissile bond between the N-extein and intein is in an unusual conformation. Interestingly, we find that the Pyrococcus horikoshii RadA mini-intein, which naturally lacks the HEN domain and is highly conserved in sequence compared to TkE, is not prone to NTC. Additionally, we find that the reintroduction of the HEN domain does not prevent NTC. Our results provide an alternative intein-based system – that does not require either an external nucleophile or prolonged incubation at high temperature (>50°C) to stimulate NTC – that controls intein activity within a temperature range amenable to most mesophilic experimental organisms.
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Sigma Xi Poster Competition
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 activity are of considerable interest. Here, we characterize a variant of the Thermococcus kodakarensis RadA intein with the homing endonuclease (HEN) domain deleted that can perform controllable protein splicing and N-terminal cleavage (NTC) in the mesophilic temperature range. We find that intein activity is largely inhibited during expression in Escherichia coli at low temperature (15C) and activated upon a slight temperature increase (>20C). We observe a differential response to the external nucleophiles hydroxylamine and dithiothreitol, which suggests that the scissile bond between the N-extein and intein is in an unusual conformation. Interestingly, we find that the Pyrococcus horikoshii RadA mini-intein, which naturally lacks the HEN domain and is highly conserved in sequence compared to TkE, is not prone to NTC. Additionally, we find that the reintroduction of the HEN domain does not prevent NTC. Our results provide an alternative intein-based system – that does not require either an external nucleophile or prolonged incubation at high temperature (>50°C) to stimulate NTC – that controls intein activity within a temperature range amenable to most mesophilic experimental organisms.