Team:Queens Canada/Project



QGEM’s 2014 project is titled “Inteins: Inspired by Nature, Accessible by Design.” Inteins are self-splicing amino acid sequences within host proteins that are able to excise themselves out auto-catalytically and ligate the flanking host protein sequences, or exteins. This relatively unknown technology has huge potential in the area of post-translational modification because of its speed and modularity. QGEM is working with inteins on two main levels to show their use as a foundation tool in synthetic biology, as well as a solution to a real world problem.

Project 1: Photo-intein

Unlike inteins that are found in nature, some inteins have separate C and N terminus domains that only splice when they are in close proximity to each other. These inteins are called trans-inteins. Our photo- intein project was inspired by the work of Tyszkiewicz and Muir in 2008; they created a light-activated protein splicing system using two chromoproteins, Phytochrome B (PhyB) and Phytochrome Associated Factor 3 (PIF3) and the VMA intein, naturally found in S. cerevisiae. After splitting the VMA intein, PhyB and PIF3 were attached to the C and N termini of the intein respectively. PhyB and PIF3, which associate when exposed to 660nm light, are able to bring the N and C termini of the intein together and initiate the splicing reaction. We aim to improve upon this with another intein, the NPU intein. The NPU intein, which is found in the cyanobacterium N. punctiforme, is up to 60x faster and may be able to speed the reaction time from 5 hours to 5 minutes. This reaction speed coupled with the ability to control the process with light can give researchers a valuable new tool to control protein expression at the post- translational level.

Project 2: Mito-intein

Mitochondrial disease often stems from defective mitochondrial genes. Most mitochondrial genes have migrated to the nuclear genome, but there are still several mitochondrial proteins which are coded by the mitochondrial genome. When the mitochondrial genes are mutated and unable to be rescued by the nuclear genome, mitochondrial disease can arise. Several studies have attempted to allotopically express mitochondrial genes in the nucleus and transport them back into them mitochondria, but we are the first to attempt this with inteins. Using a trans-intein, we will try to create a proof-of-concept project that shows the ability of inteins to transport proteins into the mitochondria. We are using a split GFP protein, attaching each portion to a trans-intein part. By attaching mitochondrial targeting sequences (MTS) to each portion of the trans intein and its respective split GFP part, we will inhibit intein splicing until the immature peptide is transported to the mitochondria and the MTS is cleaved off. At this point, the intein’s N and C termini will associate and splice out, bringing together the GFP fragments; at this point, the GFP will fluoresce. With this project, we hope to show the applicability of inteins in medicine and disease.