Expression of Microcin J25 in Yeast

Abstract

Peptides help bridge the gap between small molecule drugs (< 500 Da) with high oral bioavailability and large biologics (> 5000 Da) with high specificity, but have the challenge of oral bioavailability due to degradation by proteases. Lasso peptides, however, have a unique structure that is resistant to such degradation. Microcin J25 (MccJ25) is a peptide inhibitor that targets the RNA polymerase (RNAP) of Gram-negative bacteria and has a specific structure that is strengthened by a steric lock. Harvesting large quantities of MccJ25 in vivo proves difficult due to either export of the peptide from the cell or, when export is inhibited, toxic buildup of the RNAP-targeting peptide. This research aims to express MccJ25 in yeast rather than bacteria to avoid this issue of toxicity. Through the introduction of yeast promoters to the MccJ25 gene sequence, this work sought to construct a unique plasmid containing the bacterial MccJ25 genes and associated yeast promoters via utilization of a unique restriction enzyme mechanism (using MreI and XmaI). This work also aimed to compare the expression of MccJ25 in yeast to that in E. coli to determine whether significantly larger quantities of MccJ25 can be harvested from yeast. Yeast promoters were able to be introduced to the bacterial genes, but usage of the unique restriction enzyme pair to construct the final plasmid failed. Further experiments will be done in an attempt to construct this plasmid and test its expression in yeast.