Engineering spatial segregation within bacterial hosts for bio-therapeutic protein production
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
The production of bio-therapeutic proteins of industrial interest in recombinant hosts is becoming an increasingly popular alternative to production in mammalian cell culture which can be slow and expensive. The overall aim of this project is to develop a bacterial system for recombinant protein production by engineering compartmentalisation within the cytoplasm of E. coli which may allow for an environment that promotes production and purification of post-translationally modified proteins. In nature, compartmentalisation is achieved by localising enzymes inside of sophisticated proteinaceous organelles called bacterial microcompartments (BMCs). In the past decade, significant progress has been made to engineer these BMCs, to permit the construction of synthetic microcompartments within the bacterial cell. In this project the potential of these structures to be used for the production and purification of recombinant proteins is explored.
The project will expand our understanding of the fundamentals of engineering synthetic compartmentalisation, the benefits and limitations of protein compartmentalisation and inform the rational design of future microcompartment-derived technologies.
The project is aligned with the EPSRC centre for Doctoral Training in Bioprocess Engineering Leadership. It provides new synthetic biology approaches to the redesign of cellular chemistry and will impact in the development of cost-effective and sustainable bio-based manufacture.
The project will expand our understanding of the fundamentals of engineering synthetic compartmentalisation, the benefits and limitations of protein compartmentalisation and inform the rational design of future microcompartment-derived technologies.
The project is aligned with the EPSRC centre for Doctoral Training in Bioprocess Engineering Leadership. It provides new synthetic biology approaches to the redesign of cellular chemistry and will impact in the development of cost-effective and sustainable bio-based manufacture.
Planned Impact
The IDC has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for new venture creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to wider society via involvement in public engagement activities and encouraging future generations of researchers.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
Organisations
| Description | Bacterial Microcompartments (BMCs) are considered bacterial organelles, as they contain metabolic pathways inside. They are found naturally in approximately 20% of bacterial genomes and have an icosahedral shape, constituted by pentamers and hexamers completely composed by proteins. BMCs confine specific metabolic pathways in order to prevent the diffusion of toxic or volatile products into the cytoplasm of bacteria that possesses them. For this project, Escherichia coli (E. coli) alkaline phosphatase (PhoA), a widely used and well characterized model protein that requires its two disulfide bonds in order to be functional was used as an object of study, and in some conditions, PhoA was tested in the presence of Erv1p, a Saccharomyces cerevisiae S288c FAD-dependent sulfhydryl oxidase, which is an active catalyst of disulfide bond formation in the mitochondrial intermembrane space and essential to maintain the cysteine residues in an oxidized state. As BMCs are proteinaceous capsules, it was thought that, by encapsulating non-native proteins with disulfide bonds, these would be protected from the reducing pathways present in the bacterial cytoplasm and BMC shells would provide an environment that allowed the correct folding of proteins requiring disulfide bonds. PhoA was directed into the BMCs, and it has been confirmed that this protein is in fact encapsulated into BMCs, as per Transmission Electron Microscopy analysis. However, the encapsulation efficiency is low when co-expressed from a different plasmid than a plasmid expressing BMC shells. It has been observed that the encapsulation efficiency increased when co-expressing PhoA and BMC shells from the same plasmid. The same observations are valid for Erv1p encapsulation, although the encapsulation efficiency is slightly higher in both cases (co-expressing Erv1p from a different plasmid than BMC shells, and co-expressing Erv1p from the same plasmid as BMC shells). BMC shell does not seem to help PhoA's enzymatic activity, either because the BMC does not shield PhoA from the reducing pathways or because PhoA is encapsulated already misfolded. PhoA's enzymatic activity improves slightly when co-expressed with Erv1p. This co-expression is not improved in the presence of BMC shells. Expressing the proteins of interest (POI) to be encapsulated into the Bacterial Microcompartments (BMCs) from the same plasmid from where the BMC shell proteins are expressed from increases the efficacy of encapsulation. After designing a new construct with similar characteristics to the original Pdu BMC genome, it has been found that little to no aggregates are formed. Instead, rose/like shapes are formed, indicating that proteins are not as insoluble as when produced from the synthetic plasmids used before in this project. |
| Exploitation Route | Currently, incorrect protein folding is one of the major pharmaceutical industry challenges where protein production carries a high cost1. Disulfide bonds are a special kind of covalent bond formed in an oxidizing environment, and these bonds are essential for enzymes to function correctly. This project aims to develop an alternative method to traditional disulfide bond formation approaches by engineering compartmentalisation within the bacterial cytoplasm, such as Escherichia coli (E. coli), to provide an environment that promotes correct protein folding and disulfide bond formation of recombinant proteins. If the outcome proves successful, we are looking to enable better folding and stability for biotherapeutic target proteins (i.e. Granulocyte Colony Stimulating Factor (GCSF) and antibody fragments), and investigate the feasibility at which these folding systems can be produced in industrial hosts to aid product purification at low costs. |
| Sectors | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |