Development of an integrated system for the production and delivery of recombinant biotherapeutics

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

The bacterium E. coli is a standard host for making commercially relevant proteins e.g. recombinant insulin. However, there are limits to the types of protein that can be produced in E. coli. As most of these proteins are not derived from E. coli and are produced at levels much greater than E. coli would normally produce any natural protein, the E. coli bacterium often deals with them by storing the proteins in an inactive form in a compartment of the cell called the inclusion body. The accumulation of proteins in inclusion bodies causes problems for commercial production, including expensive steps to recover the proteins in an active conformation. To overcome these problems some proteins are produced in another compartment of the cell called the periplasm. However, if too much protein is made in the periplasm it can burst the cell open, killing the cell in the process, and therefore little or no protein is produced. Even those that are stable when produced in the periplasm will need expensive purification as there are many other native E. coli proteins in this part of the cell. When expressing a non-native protein the best place for the E. coli bacterium to place the protein is outside of the cell - this prevents accumulation of the protein in inclusion bodies or in the periplasm which might lead to cell death. In addition, the laboratory strains of E. coli produce none, or very little, native proteins outside of the cell. Thus, when proteins are produce outside the cell they are relatively clean and need little cleaning up. However, for a number of technical reasons it has proved to be very difficult to produce proteins outside the bacterial cell. Here we will utilise a very simple system called the Autotransporter system which has a proven track record for producing non-native proteins outside of the cell. We will attempt to optimise the system for a number of commercially relevant proteins.

Technical Summary

Gene cloning and expression rarely limit the production of recombinant proteins required for the production of biopharmaceutical products, but substantial bottlenecks arise from protein folding, post-translational modifications and secretion. Against this background, it is too often assumed that product recovery and downstream processing present minor technical problems that current technology can overcome. Recent advances in chromatographic techniques make such an assumption reasonable, but only if the protein can be delivered from the fermenter in a soluble form. The primary aim of this proposal is therefore to design a general strategy for the accumulation and secretion of biopharmaceutical proteins. The critical innovative steps will be to exploit three unique properties of autotransporters: their ability to secrete themselves out of bacteria into the surrounding culture fluid; their ability to co-transport partner proteins, for example, a desired biopharmaceutical protein, by a piggy back mechanism; and finally their ability to release the transported protein from the autotransporter itself. In this application we have three primary aims (1) We will specifically determine the components of the autotransporter system that are essential to achieve extracellular release of target proteins (2) We will optimise the system for maximal protein production using traditional microbial physiology techniques. At this juncture, the two major requirements for downstream processing are a dewatering step; and the separation of the desired product from the major contaminant, the bacterial cell. (3) We will apply knowledge from the first two aims to optimise a vaccine strain of Salmonella for delivery of biopharmaceutical proteins in vivo.

Publications

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Beatson SA (2011) Genome sequence of the emerging pathogen Aeromonas caviae. in Journal of bacteriology

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Flores-Langarica A (2011) T-zone localized monocyte-derived dendritic cells promote Th1 priming to Salmonella. in European journal of immunology

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Marshall JL (2012) The capsular polysaccharide Vi from Salmonella typhi is a B1b antigen. in Journal of immunology (Baltimore, Md. : 1950)

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Ross EA (2012) Thymic function is maintained during Salmonella-induced atrophy and recovery. in Journal of immunology (Baltimore, Md. : 1950)

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Ross EA (2011) CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection. in Journal of immunology (Baltimore, Md. : 1950)

 
Description House of Parliment Infection research awareness
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Industrial ITN
Amount £850,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2013 
End 01/2017
 
Title imporved strains for protein production 
Description improved strains fo rprotein production 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact patent protection granted 
 
Title Protein Secretion 
Description A bacterial expression construct comprises a nucleic acid sequence encoding a secretion unit peptide comprising less than 300 amino acids of the C-terminus of a SPATE-class bacterial autotransporter polypeptide, the secretion unit peptide 5 comprising: (i) the a-helix; (ii) linker; and (iii) ß-barrel region of the ß-domain of the autotransporter polypeptide. Such an expression construct, and associated nucleic acids and peptides, find application in the expression of proteins of interest from a host bacterial cell to the cell culture medium. 
IP Reference WO2012110821 
Protection Patent application published
Year Protection Granted 2012
Licensed No
Impact Ongoing development with Industrial partners
 
Title Recombinant protein production 
Description Filed patent covering exploitation of this research for recombinant protein production 
IP Reference US7470523 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact This teachnology has been made freely available to the community. It is currently being trialed in industry
 
Title bioprocess 
Description bioprocess 
Type Support Tool - For Medical Intervention
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact award of a personal fellowship 
 
Company Name Prokarium 
Description Spin-out company developing vaccines 
Year Established 2011 
Impact company based on live attenuated vaccines
 
Description Presentation for KTF 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation for training individuals in knowledge transfer

Award of a personal fellowship
Year(s) Of Engagement Activity 2009
 
Description The Guradian Newspaper weekly science Podcast 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact podcast which sparked high levels of p[ublic attendance at the monthly public science talk Cafe Scientifque in Birmingham

none
Year(s) Of Engagement Activity 2010