A Protein Production Facility for Biomedically Driven Structural Proteomics

Lead Research Organisation: University of Oxford
Department Name: Structural Biology

Abstract

The human genome project, over some 10-15 years, has advanced by many orders of magnitude DNA sequencing and the manipulation and management of large biological data bases. The resulting sequence of the human genome has been hailed as an epoch making achievement ushering in as yet largely uncharted opportunities for advances in human healthcare. At a pragmatic level it is generally accepted that such advances can only occur when the genomic information is complemented by advances in our understanding of protein function. A full functional description of a protein requires knowledge of its 3D structure, often as part of a large multi-molecular assembly, but the rate of determination of genomic sequences has far outstripped that of structure determination. The aim of the Oxford Protein Production Facility is to facilitate this process by developing high throughput tools for the over-expression and structure solution of human proteins and proteins from human pathogens. In the pilot phase the project has assembled a pipeline for protein production involving sophisticated data management and robotic technologies in several areas. These technologies are highly effective for proteins from bacterial pathogens and the challenge now is to refine these technologies to increase their effectiveness for human and viral proteins. Target selection will involve choosing areas where problems of biomedical importance can be advanced more effectively than by the application of standard laboratory methods. The intention is to mesh with well established networks of interdisciplinary research covering molecular, cellular and clinical studies. We have identified target areas addressing Niesseria proteins, viral proteins, human immune proteins and proteins relevant to cancer.

Technical Summary

Future funding of the OPPF will be used to achieve three major goals. Firstly, to maintain UK structural biology at the fore front of technology development in high throughput protein production and crystallisation. Secondly to apply this technology to challenging biomedical problems through the development of a portfolio of collaborative projects. Thirdly to make current and future technology available to the wider biology community through training opportunities. These aims are obviously not mutually exclusive and to a large extent will be addressed together.

COST MODEL: The costs in the electronic form represent the full costs of the OPPF project. However the consumable costs required from the MRC will be less since we will implement a cost recovery model, details of which are given in the research proposal.
Implementing this model produces a total projected cost to the MRC of #2,476,816 for the first 2 years and #5,041,564 for the total 4 years.

Publications

10 25 50
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Albeck S (2006) SPINE bioinformatics and data-management aspects of high-throughput structural biology. in Acta crystallographica. Section D, Biological crystallography

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Alzari PM (2006) Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. in Acta crystallographica. Section D, Biological crystallography

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Aricescu AR (2006) Eukaryotic expression: developments for structural proteomics. in Acta crystallographica. Section D, Biological crystallography

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Assenberg R (2008) Expression, purification and crystallization of a lyssavirus matrix (M) protein. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Au K (2006) Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis. in Acta crystallographica. Section D, Biological crystallography

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Au K (2008) Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (L-Ala-P). in Acta crystallographica. Section F, Structural biology and crystallization communications

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Berrow NS (2006) Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. in Acta crystallographica. Section D, Biological crystallography

 
Description MRC collaborative grant
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Project grant/Meningitis UK
Amount £150,000 (GBP)
Organisation Meningitis Now 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Title Cloning 
Description Various expression vectors produced and antibody production method established (seePathology Collaboration for latter) 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact 18988019 18662785 17317681 Above are pubmed ids for methods papers. Vectors have led to many results papers, which I dont have details of. 
URL http://europepmc.org/abstract/MED/18988024
 
Description Fab Cloning and Expression 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have introduced a generic method for the production of antibody fragments
Collaborator Contribution Joint development
Impact Methods reported in 18662785 and Methods in Molecular Biology (in press)
Start Year 2006
 
Description Insect Cell Expression 
Organisation Oxford Brookes University
Department School of Life Sciences Oxford Brookes
Country United Kingdom 
Sector Academic/University 
PI Contribution Testing and developing methods with Professor Linda King (Oxford Brookes University and Oxford Expression Technology Ltd)..
Collaborator Contribution Help in developing methods for insect cell expression
Impact Three publications in peer-reviewed journals: 19655260, 18781697, 20441568
 
Title pOPIN vectors 
Description Vectors produced and distributed via MTAs 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed No
Impact Various structures