Working Group 1Planing 1st Semester 2013

Planing Overview

• General Plan

• Tasks Description • Deliverables: 1st Semester 2013

• WG1: Recruiting Groups

Planing Overview

Non-characterized Proteins

Recombinant Proteins

Planing Overview

Non-characterized Proteins

Recombinant Proteins

Universidad de Salamanca-CSIC

Clones Collection ( 9800)

Clones Collection

Clones Collection ( 9800) Clones Collection ( 13500)

13500 clones ( 2013-2014)

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 ?)

In situ Protein Expression

• IVTT: Rabbits vs HeLA

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)

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

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

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 $$$ Other sources $$$$

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)

Now is time for

• Suggestions • Recommendations • Questions• Considerations • More groups in WG1• etc…..

Work flow between WP 1 and WP-MRM -Deliverable Options: Data Protein Gel band IVTT-system Protocols -Protein validated by three labs

• 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

Scouting

• 500 Euros/protein for 200 ug• 200 Euros/protein full process (20 ug

estimated)• 300 proteins total cost= 60000 Euros/2 years

•

Antibodies

http://dnasu.asu.edu/DNASU/GetVectorDetail.do?vectorid=157