Industrial Biotechnology Centre
Research and Areas of Expertise
School of Biosciences
The School of Biosciences at the University of Kent has a long history of delivering high quality research and training across the Bioprocessing remit. This has resulted in the output of personnel trained in diverse areas of bioprocessing, high quality research publications, and IP that is used for novel manufacturing approaches in recombinant protein bioprocessing. Central to this continued success has been the recognition that research and training in bioprocessing will provide a greater understanding of the biology underpinning advances in bioprocessing. Projects have been designed to work with funding agencies, academic colleagues and industrial partners to deliver research and training in areas of strength at Kent that help meet the current strategic need of the industrial and academic bioprocessing community.
Recombinant protein expression systems
Microbial Cell Engineering
Molecular Processing Science
Professor William Gullick
Professor Mark Smales
Professor Mick Tuite
Professor Martin Warren
Dr Anthony Baines
Dr Mark Howard
Dr Peter Klappa
Dr Dan Lloyd
Dr Peter Nicholls
Dr Richard Williamson
Dr Gary Robinson
Dr Marcus Allen
Dr Martin Carden
Dr Tobias von der Haar
Dr Dan Mulvihill
Dr Campbell Gourlay
Technologies available for licensing
Improved protein secretion - Professor Mick Tuite
Scientific Objectives and Themes
Molecular Processing research and training at Kent is initially focussed upon our strengths, investigating the mechanisms and cellular requirements that influence disease states and the synthesis of therapeutic agents and biomedicines from biological systems (e.g. bacterial, yeast, mammalian systems). The initial scientific objectives and themes of work in the Centre from Biosciences are:
Theme 1 - Investigate and determine the cellular constraints that limit the production of biotherapeutic medicines...
Investigate and determine the cellular constraints that limit the production of biotherapeutic medicines from prokaryotic and eukaryotic expression systems and, to develop experimentally verifiable predictive models based upon these data.
The scientific objectives within Theme 1 will be to investigate the cellular constraints that limit or enhance the expression of therapeutic protein molecules from bacterial, yeast and mammalian expression systems. The data from these studies will then be used to develop predictive models that work at either the molecular (predicting the impact of molecular characteristics on processing decisions, performance and product properties), cellular (the effect of perturbations of cellular characteristics and the environment on processing efficiency), or industrial scale levels with a view to enhancing the yields, activity and quality of therapeutic biomolecules and medicines. In doing this the following aspects will be focussed upon:
(a) mRNA translation and its control during molecular processing in yeast and mammalian cell systems;
(b) Determination of limitations on cell growth and cell specific productivity and engineering strategies to improve these;
(c) Analysis of constraints throughout the gene expression pathway to define limitations in molecular processing of biomolecules;
(d) Design of biomolecules for more efficient molecular processing and enhanced efficacy;
(e) Gene expression and control with respect to perturbation of the cell environment and cell signalling and the effect on molecular processing of biomolecules;
(f) Understanding and preventing aggregation of both cytoplasmic and secretory proteins.
Theme 2 - Determine how cells respond to protein aggregation and to identify cell engineering strategies...
Determine how cells respond to protein aggregation and to identify cell engineering strategies that may be used to evade the problems protein misfolding presents both to the organism/expression system and downstream processing.
From the expertise in Biosciences theme 2 aims to determine how cells respond to protein aggregation and to identify cell engineering strategies that may be used to evade the problems protein misfolding presents. All eukaryotic cells contain a finely balanced quality control system to ensure their newly synthesised proteins are correctly folded. At the heart of this QC system lies an effectively co-ordinated and optimised protein chaperone network. When protein misfolding occurs, these errors are usually effectively reversed where possible through this network. Alternatively, the misfolded protein is disposed of via the ubiquitin-proteasome pathway. Critically, protein misfolding often triggers protein aggregation that not only leads to a ‘loss-of-function’ of the aggregated protein, but also a gain-of-function’ through the formation of toxic oligomeric species formed through an ‘off pathway’ step. It can also impact on ‘protein homeostasis’ by triggering other proteins to misfold. Protein aggregation undoubtedly has a major impact on the fitness of a cultured eukaryotic cell or can lead to disease at the organismal level. This theme will focus upon the following scientific aspects:
(a) Design and exploitation of cellular systems for optimum and authentic protein folding. Crucial to the exploitation of eukaryotic expression systems, which in our case will be CHO and yeast (Pichia and Saccharomyces), is the prevention of unwanted protein aggregation. Failure to correctly fold a native or recombinant protein will result in a loss of productivity as well as authenticity. Our strategy will be to define the components of the chaperone network that deals specifically with protein aggregates and to engineer cells to ensure such activities are able to deal effectively with cytoplasmic and secreted recombinant proteins.
(b) Development and exploitation of non-animal models of protein misfolding diseases. A series of yeast strains and cultured cell lines expressing a range of different proteins whose misfolding has been implicated in human disease, will be developed. They will be exploited both to facilitate a further understanding of the cellular responses to protein misfolding but will also provide a novel screening platform for drugs that prevent the formation of toxic, disease-associated oligomers. Early targets will be Alzheimer’s disease (A) and Amyotrophic Lateral Sclerosis (ALS; superoxide dismutase).
Theme 3 - Enhance our understanding of how biochemical pathways operate in a synthetic biology context...
Enhance our understanding of how biochemical pathways operate in a synthetic biology context, how they are controlled, and how they can be engineered to enhance the metabolic ability of the host cell.
The scientific objective of Theme 3 is aimed at increasing our understanding of how synthetic biology and biochemical pathways operate, how they are controlled, and how they can be engineered to enhance the metabolic ability of the host cell. Metabolic engineering and synthetic biology relate to the rewiring or rewriting of the genetic code, enhancing and creating things that are beyond the range of existing biology. To begin with, this may involve the engineering of a metabolic pathway into an organism in which it does not exist, or perhaps the biosynthesis of an unnatural metabolite but ultimately it may result in the construction of an entire artificial organism. The advantages of such engineering projects are clear in that they would allow the synthesis of a range of designer chemicals, vitamins, antibiotics and biofuels as well as the generation of organisms that could be used in bioremediation, detoxification processes or the sensing of toxins or explosives. The construction of molecular cell factories or engineered life forms requires a multidisciplinary approach. This new era of synthetic biology is not merely a genetic engineering challenge - nor it is an in silico theoretical aspiration for system biologists - but it does in the first instance represent a genuine opportunity to learn about cells and to apply a reductionist approach to constructing simpler metabolic circuits. The engineering of the pathway for artemisinic acid, a key precursor of a vital antimalarial drug, exemplifies the importance of the technique in tackling major disease. Engineering metabolism thus has the opportunity of bestowing favourable genetic traits and at the same time observing genome-physiology relationships making metabolic engineering an important tool in the understanding of functional genomics. Research into metabolic engineering and synthetic biology involves both strategic and applied research relating to the understanding and exploitation of biological systems. Moreover, it requires the advancement of technology (both analytical and in silico) and provides a superb training for scientists and engineers, which meet the needs of users and beneficiaries in bioprocessing, chemical, healthcare, and pharmaceutical industries, thereby contributing to the economic competitiveness of the United Kingdom and the quality of life.
The areas of focus related to the metabolic engineering theme include:
(a) The enhanced biosynthesis of vitamins in useful dietary forms;
(b) The engineering of pathways for quinones, which can be used as building blocks for a range of drugs;
(c) The elucidation of biochemical pathways for a number of natural alkaloids and their metabolic engineering into bacteria;
(d) Metabolic engineering of eukaryotic cells for enhanced protein production;
(e) Development of predictive models describing how perturbation of metabolic pathways influences cellular phenotype.<
Hayes NVL, Smales CM, Klappa P. Protein disulfide isomerise does not control recombinant IgG4 productivity in mammalian cell lines. Biotechnology and Bioengineering, in press.
Josse L, Smales CM, Tuite MF (2010) Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells. Biotechnology and Bioengineering, 105 556-566.
Masterton RJ, Roobol A, Al-Fageeh MB, Carden MJ, Smales CM (2010) Post-translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells. Biotechnology and Bioengineering, 105 215-220.
Povey JF, Perez-Moral N, Noel TR, Parker R, Howard MJ, Smales CM (2009) Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices. Biotechnology Progress, 25 1217-1227.
Al-Fageeh, M.B. & Smales, C.M. (2009) Cold-inducible RNA binding protein (CIRP) expression is modulated by alternative mRNAs. RNA, 15, 1164-1176.
Cottier, F. & Mühlschlegel, F.A. (2009) Sensing the environment: Response of Candida albicans to the X factor. FEMS Microbiology Letters, 295, 1-9.
Leadsham, J.E., Miller, K., Ayscough, K.R. Colombo, S., Martegani, E., Sudbery, P. & Gourlay, C.W. (2009) Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast. Journal of Cell Science, 122, 706-715.
Mead, E.J., Chiverton, L.M., Smales, C.M. & von der Haar, T. (2009) Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates. Biotechnology and Bioengineering, 102, 1593-1602.
Brindley, A.A., Pickersgill, R.W., Partridge, J.C., Dunstan, D.J., Hunt, D.M. & Warren, M.J. (2008) Enzyme sequence and its relationship to hyperbaric stability of artificial and natural fish lactate dehydrogenases. PLoS ONE, 3, e2042.
Nguyen, V.D., Wallis, K., Howard, M.J., Haapalainen, A.M., Salo, K.E.H., Saaranen, M.J., Sidhu, A., Wierenga, R.K., Freedman, R.B., Ruddock, L.W. & Williamson, R.A. (2008) Alternative conformations of the x region of human protein disulphide-isomrase modulate exposure of the substrate binding b’ domain. Journal of Molecular Biology, 383, 1144-1155.
Parsons, J.P., Dinesh, S.D., Deery, E., Leech, H.K., Brindley, A.A., Heldt, D., Frank, S., Smales, C.M., Lunsdorf, H., Rambach, A., Gass, M.H., Bleloch, A., McClean, K.J., Munro, A.W., Rigby, S.E., Warren, M.J. & Prentice, M.B. (2008) Biochemical and structural insights into bacterial organelle form and biogenesis. Journal of Biological Chemistry, 283, 14366-14375.
Byrne, L.J., Box, B.S., Cole, D.J., Ridout, M.S., Morgan, B.J.T. & Tuite, M.F. (2007) Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride. Proceedings of the National Academy of Sciences of the United States of America, 104, 11688-11693.
Karala, A.-R., Psarrakos, P., Ruddock, L.W. & Klappa, P. (2007) Protein disulfide isomerases from C. elegans are equally efficient at thiol-disulfide exchange in simple peptide-based systems but show differences in reactivity towards protein substrates. Antioxidants and Redox Signaling, 9, 1815-1823.
von der Haar, T., Oku, Y., Ptushkina, M., Moerke, N., Wagner, G., Gross, J.D. and McCarthy, J.E.G. (2006) Folding transitions during assembly of the eukaryotic mRNA cap-binding complex. J. Mol. Biol. 356, 982-992.
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