Engineering the bacterial flagellum: conversion into a high efficiency protein secretion machine

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Charlotte Green, M. Hicks, F. Ying, P. C. Wright and G. P. Stafford

The University of Sheffield, United Kingdom

Secretion into culture media is a desirable route for biomanufacturing proteins employing a prokaryotic host, as they will be free from cytoplasmic contaminants. In gram negative bacteria, fusion of target proteins to a classical signal peptide will simply direct undesirable periplasmic export, possibly resulting in proteolysis, complicated downstream processing or unpredictable secretion that can complicate scale up. The bacterial flagellum is not only a highly efficient nanomotor, but also an efficient secretion machine for flagella structural proteins, extruding them through the flagellar lumen before assembly at the distal growing tip. It contains several thousand monomers of flagellin (FliC), making FliC one of the most abundant extracellular proteins in E. coli. Flagellar biogenesis is extensively characterised and indicates that the flagella represents a structure that is amenable to engineering into an efficient protein secretion device. We have constructed strains with a truncated flagellar structure that we have transformed into a streamlined secretion conduit and demonstrated secretion of eukaryotic and prokaryotic proteins, by targeting with an mRNA and protein signal to the Flagellar Type III secretion apparatus. We are currently working on a number of exemplar proteins that also require manipulation of the E. coli metabolic network to increase production of proline rich eukaryotic proteins.