Abstract
The present study details the use of split inteins to generate cyclic peptides. Split inteins can be considered as protein splicing factors, capable of forming a peptide bond between two separate proteins or peptides. Split inteins have been used to join the two termini of a peptide together, generating a head-to-tail cyclised peptide, through a process called SICLOPPs (Split Intein Circular Ligation of Proteins and Peptides). Cyclic peptides are appealing potential therapeutics, however, their use has been limited due to the lack of a cost effective system able to produce large quantities of cyclic peptides. In the present study several attempts were made to develop a conditional system for the biosynthesis of cyclic peptides using thermal regulation of SICLOPPs, these attempts were not successful for various reasons. In addition to the thermal regulation of SICLOPPs, the present study explores the development of an improved biosynthesis system for the production of cyclic peptides, ULYSSIS (Universal Ligation bY a Secondarily Split Intein System). ULYSSIS builds on the existing tool, SICLOPPs. ULYSSIS is a controlled system, where peptide cyclisation occurs following isolation of the ULYSSIS precursors from an expression host. In this way ULYSSIS is used to produce cyclic peptides \textit{in vitro}, which enables the production of pure cyclic peptide products.
The present study also contains a bioinformatic analysis of DnaE split inteins from cyanobacteria, focusing on the identification of conserved motifs which may play a in role protein splicing. This analysis revealed a novel interaction which was then focused on for further analysis.