Testing a prototype engineered bacterial flagella Type III Secretion System (FT3SS) for ‘true’ protein secretion in biotechnology

Dr Graham Stafford, University of Sheffield
Fujifilm Diosynth Biotechnologies


“This project enabled FDB to understand the applicability of this new technology to the type of  products  that we produce, and to be able to compare that with current production methods.”

Dr Ian Hodgson, Fujifilm Diosynth Biotechnologies

The Challenge

Many biotechnology companies aim to develop new processes for cheap, efficient and clean production of medicinal proteins. Currently, the protein insulin is made in bacteria and is harvested by bursting the bacterial cells. The medicinal protein must then be separated from other bacterial components using expensive processing methods.

This production pathway could be simplified by developing bacteria that secrete medicinal proteins out of the cell. This would greatly reduce processing time and costs. One natural system that could be exploited for one-step secretion of medicinal proteins from bacteria is the flagellum: a long, hollow corkscrew-like filament that, when rotated, enables bacteria to swim. Bacteria also use the flagellum as a ‘pipeline’ through which proteins made inside the cell are transported into the environment. This protein secretion is fast, as each flagellum transports ~900 proteins every minute.

The Research

University of Sheffield logoDr Graham Stafford is a Reader at The University of Sheffield. His research focuses on the microbiology of bacteria, host-pathogen interactions, engineering of bacterial flagella for protein excretion, glycan harvesting enzymes and sugar transport across bacteria membranes. All this work has significant application for industrial Biotechnology.

Dr Stafford applied for a Business Interaction Voucher with Fujifilm Diosynth Biotechnologies (FDB) and Dr Gillian Fraser (University of Cambridge) to test secretion of commercially-valuable medicinal proteins through the bacterial flagellum under industrial conditions to ascertain if it has potential for further development and merits additional investment.

The teams from Sheffield and Cambridge have already modified the flagellum to optimize its function as a pipeline for direct one-step secretion of therapeutic proteins out of the bacterial cell. They have also engineered bacteria to build many short flagella ‘pipes’ and increase the protein secretion rate, and this has been tested on a small laboratory scale.

The Result

This project enabled FDB to evaluate Dr Stafford’s technology for production of industrially relevant proteins, which gave a better understanding of the potential of the technology.

The main achievement of this project was proof that this E.coli-based secretion system can export two industrially relevant Genes of Interest (GOI) supplied by FDB into culture supernatant using the strains and vectors developed in Sheffield.  Improved conditions for culture, expression and export were also established.

This project aided in the further development of the flagella system as a protein secretion vehicle with real potential to act as a one-step secretion module for protein production in biotechnology and enabled link-up with a real-world potential partner for this work.

The Future

The data proved that the system is able to produce proteins of direct IB relevance given the secretion of two proteins provided by the industrial collaborator. The IB partner will perform fermentation runs with the genetic constructs and strains produced during ongoing work in their  in-house fermentation system in the near future.

In the first instance the data will be included in a manuscript in preparation that will be submitted to a relevant IB journal in the next 2-3 months.

A full report has been provided to FDB that summarises the project – this has allowed FDB to more fully understand the potential of the system. The data from this project therefore has potential commercial impact in the biotechnology industry.

Dr Stafford and FDB are in discussion as to how to take this project forward, either via application for iCASE awards or further RCUK/ Innovate funding in the near future.

“This BIV funding allowed us to assess and improve our system with ‘real-world’ IB target proteins and produce valuable pilot data for the future.”

Dr Graham Stafford, University of Sheffield