JANICKAITE, DONATA (2017) Characterisation of single tryptophan mutants of Saccharomyces cerevisiae Ire1. Masters thesis, Durham University.
|PDF - Accepted Version|
The endoplasmic reticulum (ER) is responsible for the folding and modifications of numerous proteins produced by the cell. To ensure that only correctly folded proteins proceed to the surface, a highly conserved process called the unfolded protein response (UPR) has been developed. In mammals, signalling in the UPR is induced by membrane embedded stress sensors, IRE1, PERK and ATF6. IRE1 is the only ER stress sensor conserved across all eukaryotes.
Yeast Ire1 has a serine/threonine kinase and endoribonuclease domains needed to exert its functions. During accumulation of unfolded or misfolded proteins, Ire1 oligomerises and autophosphorylates leading to the activation of its RNase domain. The activated RNase domain then targets mRNA to remove an intron and produce a bZIP transcriptional activator (Hac 1 in yeast and XBP-1 in metazoans), which activates the downstream pathways of UPR. The aim of this study was to purify and characterise the enzymatic activity of tryptophan mutants of yeast Ire1 in order to potentially use them in future tryptophan fluorescence studies.
Three single tryptophan mutants were purified as GST-fusion proteins and characterised for kinase and RNase activity. Results presented in this thesis show one of the mutants (W855F-W1025F-Ire1) purifies well based on previously optimised protocols and two mutants probably purify as a mixture of full length and truncated proteins. All of the tryptophan mutants retain kinase activity, but only one of them has partial RNase activity (W855F-W981F-Ire1). It is also shown here that removal of the GST tag decreases the protein RNase activity, potentially because the protein becomes monomeric.
Further optimisation of the induction and purification conditions is needed to obtain full length proteins. Alternatively, it may be necessary to use a different induction system for protein purification.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Keywords:||Ire1, Saccharomyces cerevisiae, endoplasmic reticulum, unfolded protein response|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||03 May 2017 10:20|