Working Group 1 Planing 1st Semester 2013
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Transcript of Working Group 1 Planing 1st Semester 2013
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Working Group 1Planing 1st Semester 2013
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Planing Overview
• General Plan
• Tasks Description • Deliverables: 1st Semester 2013
• WG1: Recruiting Groups
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Planing Overview
Non-characterized Proteins
Recombinant Proteins
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Planing Overview
Non-characterized Proteins
Recombinant Proteins
Universidad de Salamanca-CSIC
Clones Collection ( 9800)
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Clones Collection
Clones Collection ( 9800) Clones Collection ( 13500)
13500 clones ( 2013-2014)
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Recombinant Proteins
In situ Protein expression Conventional Protein expression
•Fast ●cDNA availability●Clones sequence validated●Yield ?
•Slow ●Subcloning●Experience: E.coli, CHO, Yeast…●High Yield ( purification ?)
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In situ Protein Expression
• IVTT: Rabbits vs HeLA
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In situ Protein Expression
• IVTT: Rabbits vs HeLA
• Optimized protocols for Protein Enrichement
• MS assays ( CICBiogune, UCM, ???)
• 25 proteins expressed by HeLa IVTT ( CIC)
• 50 additional clones validated ( UCM & CIC)
• Training of HeLa IVTT at CIC ( open)
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Conventional Protein Expression
• Cloning & Subcloning strategies
• Selection of expression system: E.coli, CHO, yeast, Pichia,…)
• Non-succesfully IVTT expressed proteins • High demand of recombinant proteins by other
WGs
Preliminary Criteria
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Working Flow between WG1 & WG-MRM
• Deliverable: Data, purified protein, IVTTmix+protein, gel band, protocols,….
• Amount of protein: ~ 20 µg / Lab
• Number of Labs for MRM: 3/protein ???
• Updated list of unknown/non-characterized proteins
• Number of labs for MRM: Recruiting
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Cloning
• No in CIC collection (9800) ( 642 chr16)• No in ASU collection (13500) ( > 642 chr16?)
• Cloning & subcloning: After first year A few selected proteins Conventional cloning $$$ Other sources $$$$
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Budget Scouting
• 600 €/ Protein HeLa IVTT for 200 µg
• 250€/full process per protein ( < 20 µg)
• Milestone: 300 proteins / 2 years 60 k€ ( personal cost no included)
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Now is time for
• Suggestions • Recommendations • Questions• Considerations • More groups in WG1• etc…..
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Work flow between WP 1 and WP-MRM -Deliverable Options: Data Protein Gel band IVTT-system Protocols -Protein validated by three labs
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• 25 proteins for MRM assays-USAL+UCM+Biogune→MRM WG
• 25 novel proteins in both IVTT expression systems -USAL
• 25 for DNA sequencing to UCM_ follow-up IVTT • Heavy proteins???? Planning-options-strategy• Quantity: amount 20 ug /average per protein HeLa IVTT as first option• 300 proteínas en 2 años--milestone
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Scouting
• 500 Euros/protein for 200 ug• 200 Euros/protein full process (20 ug
estimated)• 300 proteins total cost= 60000 Euros/2 years
•
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Antibodies
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http://dnasu.asu.edu/DNASU/GetVectorDetail.do?vectorid=157