Murray State Theses and Dissertations
Abstract
Inteins, intervening proteins, are post-translational, mobile genetic elements that are translated within host proteins and removed through autocatalytic splicing. Inteins are abundant in bacterial and archaeal genomes and are usually located at sites of functional proteins necessary for an organism's survival. The locale and 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.
In these studies, we sought to elucidate specific conditions in which intein splicing and cleavage could be controlled. Here, we report an intein variant that performs controllable protein splicing and N-terminal cleavage upon a temperature shift from 15°C to >21°C. These results provide an alternative to previous systems that require an external nucleophile (e.g., dithiothreitol) or prolonged incubation at elevated temperatures (>50°C) to stimulate the reaction, controlling intein activity by temperature in a range that is useful for many experimental organisms.
Additionally, we wanted to further characterize our intein variant, TkΔE-NATA. With alanine substitutions in place of conserved threonine and cysteine residues, this intein mutant was able to undergo N-terminal cleavage. The residue right before the intein, also known as the -1 position, seemed to influence the rate of N-terminal cleavage. We tested this speculation by supplementing the -1 residue of TkΔE-NATA with one of the 20 possible amino acids and measured the rate of N-terminal cleavage. Cleavage assays were completed by incubating reactions at 37°C at varying time points for each of the 20 variants. Results show that certain residues at the -1 position affect the rate at which N-terminal cleavage occurs.
Year manuscript completed
2023
Year degree awarded
2023
Author's Keywords
Intein; Protein splicing; Protein purification; Protein cleavage
Thesis Advisor
Christopher W. Lennon
Committee Member
Gary T. ZeRuth
Committee Member
Chris Trzepacz
Document Type
Thesis - Murray State Access only
Recommended Citation
McNeal, Taylor, "Conditional Protein Splicing and N-terminal Cleavage Activated by Mild Temperatures" (2023). Murray State Theses and Dissertations. 301.
https://digitalcommons.murraystate.edu/etd/301