The Oxford Protein Production Facility-UK
Lead Research Organisation:
University of Oxford
Department Name: Structural Biology
Abstract
Proteins are essential components of all living cells. They comprise chains of amino acids coded for by specific DNA sequences and fold into three dimensional structures, that determine their function, for example as biological catalysts. Therefore, understanding protein structure and function is key to exploiting the information generated from sequencing of the human genome and in particular those of many human pathogens. Knowledge of individual proteins has made a major contribution to the development of a number of important drugs, e.g. Zanamivir (influenza neuraminidase inhibitor), Lapatinib (kinase inhibitor), Darunavir (HIV protease inhibitor). However, high throughput DNA sequencing continues to add to the database of known proteins at a rate that far exceeds our capacity to decipher their function. Therefore, accelerating the production of recombinant proteins is key to closing the gap between the acquisition of protein sequences and the determination of their structure and function. In response to this challenge, the Oxford Protein Production Facility will provide infrastructure to underpin the study of proteins on different length and time scales from atomic resolution (x-ray crystallography) to whole cell systems (optical microscopy).
Technical Summary
The Oxford Protein Production Facility-UK (OPPF-UK) is a National resource for protein science. The overall goal of the OPPF-UK is to provide the UK biological, chemical and biomedical communities with a world-class facility for the production of proteins for structural and functional research. The OPPF-UK has established a high throughput approach to the production of recombinant proteins using three main expression hosts (E. coli, mammalian cells and baculovirus/insect cells) which is provided as a service to the UK scientific community through a peer-review process. Research and development in the OPPF-UK will be focussed on streamlining the cloning, expression and purification of multi-protein complexes and membrane proteins using insect and mammalian cells. Methods will be rolled out as part of the user programme of the OPPF-UK. In parallel, data management systems will be enhanced to provide users with secure on-line access to all project data.
Planned Impact
Recombinant proteins play a key part in the drug discovery process, for example as components of biochemical screens for new chemical entities (NCEs), antigens for raising therapeutic antibodies and for crystallization to obtain structural information. We are contributing to projects involving the structure-based identification of NCEs, for example inhibitors of a novel metallo-beta-lactamase (NDM-1) which has been implicated in widespread antibiotic resistance of important bacterial pathogens. New knowledge will be added from these studies which may ultimately help in the development of new drugs. The OPPF-UK is developing a number of direct links with the biotech/pharma sector through consultancy and training and as a provider of protein production services. The OPPF-UK is also actively involved in helping to educate the next generation of potential research scientists. We contribute to school visits to the Rutherford Appleton Laboratory through staff giving talks about how proteins are studied in the OPPF-UK and our links with the Diamond Light Source. Finally, the OPPF-UK provides an important opportunity for staff development, since working in the OPPF-UK involves scientists in the management of multiple projects, dealing with deadlines and liaising with external users. This experience helps to develop transferable skills that are highly applicable not only to jobs in research & development but also other professions outside of science.
Publications
Ahmed A
(2019)
Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells
in Scientific Reports
Araujo-Montoya BO
(2020)
Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.
in Protein Expr Purif
Arena De Souza V
(2015)
Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1.
in PloS one
Aricescu AR
(2013)
Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.
in Current opinion in structural biology
Asiani KR
(2016)
SilE is an intrinsically disordered periplasmic "molecular sponge" involved in bacterial silver resistance.
in Molecular microbiology
Awanye AM
(2019)
Immunogenicity profiling of protein antigens from capsular group B Neisseria meningitidis.
in Scientific reports
Bayley CP
(2016)
Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity.
in Scientific reports
Beale JH
(2015)
Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport.
in Structure (London, England : 1993)
Bird LE
(2016)
Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.
in Advances in experimental medicine and biology
Bird LE
(2014)
Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors.
in Methods in molecular biology (Clifton, N.J.)
Bird LE
(2015)
Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.
in Journal of visualized experiments : JoVE
Butryn A
(2020)
Molecular basis for GTP recognition by light-activated guanylate cyclase RhGC.
in The FEBS journal
Byrne RT
(2013)
S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.
in Acta crystallographica. Section D, Biological crystallography
Ditsiou A
(2020)
The structure-function relationship of oncogenic LMTK3.
in Science advances
El Omari K
(2023)
Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.
in Communications chemistry
Hannon C
(2018)
Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor.
in Acta crystallographica. Section F, Structural biology communications
Hardman C
(2017)
CD1a presentation of endogenous antigens by group 2 innate lymphoid cells
in Science Immunology
Hardman CS
(2021)
IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent.
in Science immunology
Hu NJ
(2015)
GFP-based expression screening of membrane proteins in insect cells using the baculovirus system.
in Methods in molecular biology (Clifton, N.J.)
Itkonen JM
(2014)
Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli.
in BMC biotechnology
Kim YC
(2021)
Temperature-dependent secretion of Zika virus envelope and non-structural protein 1 in mammalian cells for clinical applications.
in Journal of virological methods
Knox R
(2013)
A streamlined implementation of the glutamine synthetase-based protein expression system.
in BMC biotechnology
Kulminskaya N
(2021)
Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.
in The Journal of biological chemistry
Lanyon-Hogg T
(2015)
Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.
in Analytical biochemistry
Lanyon-Hogg T
(2016)
Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase.
in Data in brief
Liu C
(2015)
A RANKL mutant used as an inter-species vaccine for efficient immunotherapy of osteoporosis.
in Scientific reports
Lukacik P
(2015)
High-resolution structures of Lactobacillus salivarius transketolase in the presence and absence of thiamine pyrophosphate.
in Acta crystallographica. Section F, Structural biology communications
Makena A
(2013)
Chromophore-linked substrate (CLS405): probing metallo-ß-lactamase activity and inhibition.
in ChemMedChem
Mathavan I
(2022)
Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.
in ACS medicinal chemistry letters
Mina JG
(2017)
Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.
in The Journal of biological chemistry
Morra R
(2017)
Optimization of Membrane Protein Production Using Titratable Strains of E. coli.
in Methods in molecular biology (Clifton, N.J.)
Nelson CR
(2020)
Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs.
in International journal of molecular sciences
Nettleship JE
(2015)
Transient expression in HEK 293 cells: an alternative to E. coli for the production of secreted and intracellular mammalian proteins.
in Methods in molecular biology (Clifton, N.J.)
Nettleship JE
(2019)
Overview of a High-Throughput Pipeline for Streamlining the Production of Recombinant Proteins.
in Methods in molecular biology (Clifton, N.J.)
Owens R
(2016)
Methods in integrated structural biology.
in Methods (San Diego, Calif.)
Owens RJ
(2019)
Streamlining the production of proteins for structural biology.
in Biophysical reviews
Owens RJ
(2015)
Structural proteomics: high-throughput methods. Preface.
in Methods in molecular biology (Clifton, N.J.)
Palazzo L
(2016)
ENPP1 processes protein ADP-ribosylation in vitro.
in The FEBS journal
Ren J
(2019)
The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus.
in Molecular immunology
Roatsch M
(2016)
Substituted 2-(2-aminopyrimidin-4-yl)pyridine-4-carboxylates as potent inhibitors of JumonjiC domain-containing histone demethylases.
in Future medicinal chemistry
Robinson JI
(2018)
Affimer proteins inhibit immune complex binding to Fc?RIIIa with high specificity through competitive and allosteric modes of action.
in Proceedings of the National Academy of Sciences of the United States of America
Romanello L
(2017)
Structural and kinetic analysis of Schistosoma mansoni Adenylosuccinate Lyase (SmADSL).
in Molecular and biochemical parasitology
Romanello L
(2019)
In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases.
in Molecular and biochemical parasitology
Sabaratnam K
(2019)
Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress.
in Protein science : a publication of the Protein Society
Salimraj R
(2019)
Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-ß-lactamases.
in The FEBS journal
Description | New001 Building research capacity for schistosomiasis drug discovery & development through high-content imaging & structural molecular biology studies |
Amount | £67,581 (GBP) |
Funding ID | MR/M026221/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2015 |
End | 05/2017 |
Description | Structural Biology in the RFI |
Amount | £1,649,512 (GBP) |
Funding ID | EP/S025243/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2018 |
End | 05/2020 |
Description | University of Oxford GCRF, HFCE |
Amount | £52,580 (GBP) |
Funding ID | 0006135 & 0005295 |
Organisation | Oswaldo Cruz Foundation (Fiocruz) |
Sector | Public |
Country | Brazil |
Start | 03/2018 |
End | 03/2019 |
Description | Building research capacity for schistosomiasis drug discovery and development through high-content imaging and structural molecular biology studies |
Organisation | Oswaldo Cruz Foundation (Fiocruz) |
Country | Brazil |
Sector | Public |
PI Contribution | Training in high throughput cloning and expression of proteins fro drug discovery research. Protein production and crystallization of Schistosoma proteins selected a potential drug targets. Delivery of seminar-based workshop at Oswaldo Cruz |
Collaborator Contribution | Expertise in drug screening Identification of potential inhibitors for co-crystallization experiments |
Impact | Technology transfer to Oswaldo Cruz. Novel results which will be published in due course. |
Start Year | 2015 |
Description | School visits |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Practical workshop for year 12 school groups ( approx. 50/visit ) on how recombinant proteins are produced using Green fluorescent protein as the example. |
Year(s) Of Engagement Activity | 2014,2015,2016 |
Description | Schools visit |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Hands-on practical workshops on proteins and protein crystallization |
Year(s) Of Engagement Activity | 2017 |
Description | Science Up Close: Rutherford Appleton Laboratory Open Day |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Hands-on crystallizing a protein for children as part of a stall about proteins and how we study them visited by attendees of the Rutherford Appleton Laboratory Open Day |
Year(s) Of Engagement Activity | 2015 |
Description | Science and Society Lecture |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Presented at the annual Science and Society lecture - talk sparked questions and discussions afterwords. Talk could lead to potential collaboration opportunists. |
Year(s) Of Engagement Activity | 2014 |
Description | Youtube video |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Video for general audience on protein production and crystallization produced by Backstage Science for the STFC. |
Year(s) Of Engagement Activity | 2012 |
URL | https://www.youtube.com/watch?v=joWNZDBqrlQ |