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The MRC Oxford Protein Production Facility - towards large scale protein production

The MRC Oxford Protein Production Facility - towards large scale protein production. Dave Stuart NIH March 2001.

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The MRC Oxford Protein Production Facility - towards large scale protein production

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  1. The MRC Oxford Protein Production Facility - towards large scale protein production Dave Stuart NIH March 2001

  2. Structural Genomics in BritainHistorically the UK is strong in structural biology, but there is little coordinated activity in structural genomics.We are way behind!- Coordination meeting 11-Dec 2001 at the Royal Society, LondonOngoing:- Wellcome Trust / Industry: SGC?- Oxford: MRC funded OPPF- Daresbury: NWSGC- The European perspective: SPINE

  3. Diamond – the new UK synchrotron • 300 M US dollars initial build costs • Joint project: Office of Science and Technology, Wellcome Trust, France • Science: • roughly 50/50 Biology/Physical Sciences • 3 out of 7 day one beamlines for HTP PX • - this commitment of resources to biology • is unprecedented – and assumes SG!

  4. Extant UK synchrotron resources • SRS: new MAD beamline under construction • ESRF: the UK own and run BM14 • MAD beamline – will be test-bed • for automation • ESRF to build HTP public PX beamline

  5. Oxford Protein Production Facility (OPPF) • First substantially funded UK structural genomics activity • MRC funding, 3 years in first instance (~8 M US dollars) • ‘Pilot project’ for larger scale activity associated with the new Diamond synchrotron • Present status: 6 full time staff • – hope to start real activity April 2002

  6. OPPF management group Dave Stuart (chair) John Bell Iain Campbell Simon Davis Robert Esnouf Jon Grimes Karl Harlos Louise Johnson Yvonne Jones Ian Jones (Reading) David Kerr Tony Monaco Gavin Screaton Dave Stammers

  7. Aims of Oxford Protein Production Facility • Link in with existing biomedical research programmes which are already using, for instance, microarray and SAGE technologies • Targets mainly human proteins relevant to human health, plus human viruses • Establish protein expression in bacteria/insect/mammalian systems • Provide protein as a resource for functional studies and structural studies (also e.g. use GFP to track protein expression) • Proteins will be ‘reagents’ for programmes aiming to look at assemblies of several components • Link in with NMR and cryo-EM • Target 1000 clones per year into pipeline

  8. Targets • Herpes viruses • Proteins characteristic of immune cell function • Protein modules • Zinc finger containing proteins / transcription factors • The cancer genome

  9. Nov 9th 2001: The OPPF is assembled!

  10. Building handover – Easter 2002

  11. Challenges from clone to structure • Bioinformatics. Data base construction, LIMS integration. • Protein expression/purification. Standardization to pipeline 1000 target proteins per year. • Crystallization. Automation of screening, detection and optimization. • Data collection/phasing. Data base integration with synchrotron.

  12. Tracking and scheduling with a Laboratory Information Management System (LIMS)Virtues evident (from accounting to data mining)Effort considerable

  13. Tracking and scheduling with a Laboratory Information Management System (LIMS)Nautilus, Thermo Labsystems

  14. Barcodes • Coding symbology likely to be adopted by the OPPF: • 128C encoding 12 numeric digits • Suggested usage format of the 12 digits: • XX YYY ZZZZZZ C • XX Oganisation identifier – the OPPF would take 44 • The range 90-99 is reserved, and will be used for three digit organisation identifiers when this becomes necessary • YYY Object identifier – eg 998 for normal Greiner plates, 999 for shallow Greiner plates, 000 for people, etc. • (Gives a range of 1000 object types) • ZZZZZZ Object content identifier – a unique identifier for this item within this object set • (Gives a range of 1000000 uniquely identified items per object) • C Triple-add-triple checksum – help prevent typos

  15. Global identification • A flexible solution would be: • Coding symbology: Unspecified • Alphanumeric/Numeric: The string must start with the numeric organisation identifier (first two or three characters), otherwise no constraint is imposed • Length of encoded string: >2 • (>3 for organisation identifiers beginning 90-99) • Each organisation then provides a web-based facility to translate their own code string into the relevant documentation for each item made available to other groups. • A centrally-maintained list maps the organisation identifier (first two digits of string) to the organisation name and the URL of the documentation-providing facility

  16. Cloning issues AS WITH ALL ASPECTS WE ARE JUST STARTING! cDNA – Human/Mouse: MRC UK Human Genome Mapping Project (ORFEUS project) - Herpes virus: viral DNA ( from clinical isolates) Cloning strategy: Gateway Expression: Bacteria / insects / mammalian Tags: 1 or 2 tags plus fusion protein option via Gateway

  17. Expression & purification issues Technology: centered on Qiagen Biorobot 8000 protocols Screening: 96 well technology Expression scale-up: technology not determined (initially shaker flasks) Larger scale purification: Centre of Excellence agreement with Amersham Pharmacia QA: mass spectrometry etc Initial work with E Coli, aim to activate Baculovirus infection route within year 1 and mammalian cell route within 2 years Initial N-term His tag construct, HRV 3C protease cleavable

  18. Qiagen Biorobot 8000 • 96 well parallel protein purification – magnetic & vacuum manifold technologies • In particular Ni NTA • For larger scale protein production • Amersham Pharmacia Akta 3D (x3)

  19. Crytallisation issues Technology: 96 well sitting drop Nanolitre drop volumes Currently Greiner plates Reservoir dispensing for screens: Robbins hydra, in-house adaptations Drop dispensing: Cartesian microsys (8 head), in-house adaptations

  20. Basic Hydra: GUI driven for dispensing varied solutions

  21. Small volume crystallisation: Cartesian Technologies 8 head dispenser (down to 20nl)

  22. Cartesian mods

  23. Crystal storage/imaging issues Technology: TAP storage vault, Veeco imager Software: Imaging software, under development

  24. Photographs of crystallisation plate (Berlin structure factory design)

  25. Crystals of Igf2R grown within 20 hours in a Greiner crystallisation plate. Total drop volume is less than 200 nl. The drop was prepared by manually pipetting 100 nl of protein solution (at 12 mg/ml) to 100 nl of reservoir solution. Well dimensions are 2.1 x 2.1 mm, crystals shown are between 60 and 120 microns long.

  26. TAP 10,000 plate storage robot Imaging: Integrated Veeco system 1 tray: 96 images / minute

  27. SPINE Structural Proteomics IN Europe

  28. SPINE • 3 Year grant – 13.7 M Euros • Technologies development: • cloning and expression, through to • crystallography and NMR • Biomedical targets: • Human pathogens: • Bacterial: TB & Campylobacter • Viral: Herpes viruses & enzyme targets • -Human proteins: • Cancer related targets • Neurological development/disorders

  29. SPINE • 11 ‘node’ responsibles: • Oxford • Stockholm • Weizman • Hamburg • Utrecht • Grenoble • York • EBI • Marseille • Strasbourg • Munich • - plus a number of other major players

  30. SPINE Also with links to small-medium companies for technology development and to most European synchrotrons

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