A Protease-Based System to Release Protein from the Surface of Escherichia coli

Authors

  • Mars Wichmann-Young University of Ottawa, Ottawa, ON, Canada
  • François-Xavier Campbell-Valois University of Ottawa, Ottawa, ON, Canada
  • Kyle Tomaro University of Ottawa, Ottawa, ON, Canada

DOI:

https://doi.org/10.18192/osurj.v5i2.8058

Abstract

We are developing a novel secretion system that uses two autotransporter constructs, which self-transport their passenger domain cargo across the outer membrane, to transport and cleave a protein of interest (POI). The tobacco etch virus protease (TEVp) is transported to the surface by the E. coli autotransporter protein YfaL. Here, it cleaves a POI from a second autotransporter construct, facilitating its secretion. Currently, premature cytosolic cleavage prevents efficient secretion. In previous work, the araBAD promoter system and theophylline riboswitch were added to allow the POI to reach the cell surface before induction of the TEVp construct. While this strategy yielded a tightly inducible system, it did not prevent premature cleavage. Therefore, we sought additional strategies to reduce cytosolic cleavage.
In this project, we implemented solutions to this challenge by controlling cytosolic expression, stability, and activity. First, the TEVp construct was expressed in a lower copy plasmid, pUdO4s. From this, we first added a C-terminus proteolysis tag to reduce the half-life of the protein. In E. coli, this tag is added to truncated proteins by the ssrA transcript, facilitating recognition and degradation by intracellular proteases. This reduces cytosolic stability and concentration, favouring surface-bound TEVp.
We expressed these constructs in E. coli and measured relative protein expression and presence of cleavage products in comparison to previous work. As expected, we observed a significant reduction in the expression of membrane-bound TEVp in the new constructs. While premature cleavage still occurred in the original construct, presence of cleavage products was minimal in the current strain, NEBExpress Iq, and little to none was present pre- or post-induction in either novel construct. This warrants further investigation into factors influencing the formerly observed premature cleavage, as well as the apparent lack of adequate cell surface-level protease activity.

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Published

2026-06-17

Issue

Vol. 5 No. 2 (2026)

Section

Undergraduate Science Research Opportunity Abstracts

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