Splicing Rates of Inteins from Thermococcus Kodakarensis
Academic Level at Time of Presentation
Junior
Major
Biochemistry & Biology
Minor
Music
List all Project Mentors & Advisor(s)
Dr. Christopher Lennon
Presentation Format
Poster Presentation
Abstract/Description
Intervening proteins, or inteins, are protein sequences translated within a host protein that excise themselves in a self-mediated splicing reaction, leaving behind a complete and uninterrupted host protein as well as a small intein fragment. Inteins can be found throughout the microbial world and are often located within essential proteins involved in DNA replication, recombination, and repair (RRR).
The archaeon Thermococcus kodakarensis is a hyperthermophilic deep-sea microbe housing 15 unique inteins across 12 proteins. Here we demonstrate the splicing rates and efficiencies of these inteins in vitro using a fluorescent reporter. Each intein was inserted in between a maltose binding protein (MBP) and a green fluorescent protein (GFP). Plasmids coding for the inteins were then transformed into Escherichia coli for expression. the MBP-Intein-GFP (MIG) reporter was purified and incubated at 59°C with timepoints taken every 10 minutes.
Using our MIG reporter and SDS-PAGE, we found the splicing rates of the 15 inteins from T. kodakarensis greatly vary. Interestingly, a few inteins—namely PolD and RFC—showed minimal splicing, but are present within essential proteins in T. kodakarensis, suggesting different conditions are required for intein splicing. Future work will explore various conditions to find factors that influence splicing of PolD and RFC inteins.
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Sigma Xi Poster Competition
Splicing Rates of Inteins from Thermococcus Kodakarensis
Intervening proteins, or inteins, are protein sequences translated within a host protein that excise themselves in a self-mediated splicing reaction, leaving behind a complete and uninterrupted host protein as well as a small intein fragment. Inteins can be found throughout the microbial world and are often located within essential proteins involved in DNA replication, recombination, and repair (RRR).
The archaeon Thermococcus kodakarensis is a hyperthermophilic deep-sea microbe housing 15 unique inteins across 12 proteins. Here we demonstrate the splicing rates and efficiencies of these inteins in vitro using a fluorescent reporter. Each intein was inserted in between a maltose binding protein (MBP) and a green fluorescent protein (GFP). Plasmids coding for the inteins were then transformed into Escherichia coli for expression. the MBP-Intein-GFP (MIG) reporter was purified and incubated at 59°C with timepoints taken every 10 minutes.
Using our MIG reporter and SDS-PAGE, we found the splicing rates of the 15 inteins from T. kodakarensis greatly vary. Interestingly, a few inteins—namely PolD and RFC—showed minimal splicing, but are present within essential proteins in T. kodakarensis, suggesting different conditions are required for intein splicing. Future work will explore various conditions to find factors that influence splicing of PolD and RFC inteins.