Examining the potential for periplasmic volume manipulation and its influence on Sec and FT3SS based protein production

Dr Graham Stafford, University of Sheffield
Fujifilm Diosynth Biotechnologies

 


“It is always a pleasure to host Undergrad students on summer projects, I know it kick-started my career and I gained great value from the experience.”

Graham Stafford, University of Sheffield


The Challenge

University of Sheffield logoE. coli is a vital organism to industrial biotechnology and is currently used for the production of a range of important biopharmaceutical proteins, such as insulin. Recent work has suggested that the volume of the periplasmic space may be important in protein production in E. coli and that increasing periplasmic volume may alleviate envelope stress responses resulting from protein production.

The aim of this summer vacation studentship was to modify the E. coli periplasmic volume by altering the length of Braun’s Lipoprotein (Lpp) and assess what effect this has on protein production in the periplasm (Sec-dependent secretion) and on one-step FT3SS-mediated secretion.

The Research

Dr Graham Stafford is a Reader in Microbiology at the University of Sheffield. The research in his laboratory focuses mainly on capitalising and using molecular microbiology knowledge to combat disease pathogens or for biotechnology applications.

Fujifilm Diosynth Biotechnologies (FDB) is a world leading company involved in the development and production of biologics, vaccines and advanced therapies.

Dr Stafford applied for a CBMNet Vacation Scholarship to fund an undergraduate student to carry out research in his laboratory. The project aimed to engineer E. coli to improve IB processes for protein production in bacteria.

The Result

In order to alter the periplasmic space of E. coli, the vacation student successfully generated a deletion strain that no longer expressed Lpp. The next step was to design and synthesise plasmids that would express mutant Lpp genes with altered lengths, in order to complement the deletion strain. These plasmids have been successfully generated.

As a model, a protein of interest to Fujifilm Diosynth Biotechnology was used to test expression in the systems under study.

Despite not completing every aim the student (Jack Hales) gained valuable experience of molecular biology, visiting the Fujifilm Diosynth Biotech facility and being embedded in a University research department.

The Future

The next steps in this project will need to generate E. coli strains that express lengthened or shortened Lpp proteins and to test these strains for protein production. It will be interesting to see how periplasmic volume affects protein production via the two secretion pathways and if increasing/reducing periplasmic volume has different effects on these.

This vacation scholarship has laid the groundwork for further research on this project and it will be taken forward by a fourth year MBiolSci student in the next year, hopefully to form the basis of further funding applications. It is also closely aligned with current research collaborations between Dr Stafford’s laboratory and Fujifilm Diosynth Biotechnologies, strengthening their academic-industrial partnership.

The undergraduate student gained some great, real-life experience in a research laboratory and also visited the FujiFilm Diosynth Biotechnologies facilities.


“I thoroughly enjoyed the project, and being part of novel research.  I also experienced the joys as well as sometimes frustrating realities of real science.”

Jack Hales, Vacation student, University of Sheffield