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Working Group 1 Mid-term Report

Working Group 1 Mid-term Report. Strategy Overview. Action PLAN Non- characterized protein Cloning & Validation Recombinant proteins Experimental design for MS assays. 16. Genes on Chromosome 16. 16.

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Working Group 1 Mid-term Report

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  1. WorkingGroup1 Mid-termReport

  2. StrategyOverview • Action PLAN • Non-characterizedprotein • Cloning & Validation • Recombinantproteins • Experimental designfor MS assays

  3. 16 Genes onChromosome 16 16 More than85 % of proteincoding genes of chromosome 16 are expressed in lymphoidcells, epitelial cells and fibroblasts.

  4. 16 Genes onChromosome 16 16 More than85 % of proteincoding genes of chromosome 16 are expressed in lymphoidcells, epitelial cells and fibroblasts.

  5. Action Plan 260 CIC collection 25 Non-characterized proteins 25 December 2012 10 August 2012 Sequencevalidated & IVTT expressed Sequencevalidated & IVTT expressed

  6. Experiments

  7. LiquidCulture LB medium + Amp 100 µg/ml + 1.5 µl glycerol stock /clon O/N, 37ºC, 300 rpm

  8. DNA Prep byPureYieldPlasmidMiniprepPromegasystem Fast High-yield Semi-automatic

  9. DNA prep MC1R TMEM170 WFDC1 LOC348174 ZNF19 SPATA2L HAGHL LRRC29 IGSF6 KCNG4 CKLFSF2 GPR114 GSG1L

  10. InsertVerificationby Xho1 & BamHIDigestion TMEM170 Marcador 1Kb WFDC1 MC1R LRRC29 KCNG4 IGSF6 CKLFSF2 GSG1L

  11. SEQUENCING ≥ 90 % sequencevalidation

  12. Recombinant Proteins • Master Library  Many libraries • Any vector can be modified to accept genes

  13. RecombinantProteins IVTT Others ( E.coli, Pichia, …)

  14. In situ ProteinExpression 168 µl DEPC water + 4 µl Leu orCys + 4 µl Met + 16 µl TnT Buffer + 4 µl RNAseout + 8 µl T7 polymerase + 0.2-2-0 1.5 µg DNA + PBS 200 µl Celllysate 90 min, 30ºC, 300 rpm 10 min, 4ºC ProteinExpressed

  15. In situ cell-free expression of non-characterizedproteins RabbitReticulocyte HeLa

  16. TNT coupledReticulocyteLysateSystem Fastproduction of RecombinantProtein Yield per reaction > 0.1-10 µg 23 of 25 non-characterized proteinsin situ expressed

  17. IVTT non-coupledfromHeLaLysateSystem High-yield In house: >100 µg / reaction

  18. Reagentsdeliveredfrom Pierce Headquarter ( Madison, WI) on 30th-Nov-2012 1of 25 non-characterized proteinsin situ expressed

  19. 1D SDS-Page Pierce expression of SPATA2L FLJ 82130 CKLPSP2 SB1X WFDC1 GSG1L SB1X SB1X AQP8 SPATA2L C16orf73 MW HSPC 105 ZNFA MW KCNG4 SB1X LOC348174 TMEM170 IRX3 LRRC29 MC1R SB1X MW HAGHL GPR114 C16orf78 DEX1 IG5F6 SB1X

  20. IVTT RecombinantProteins WB BeadsSuspensionArrays (BBAs)

  21. WB

  22. IVTT RecombinantProteins WB BeadsSuspensionArrays (BBAs)

  23. BeadsSuspensionAssays of HeLain situexpressedSPATA2L clone

  24. BeadsSuspensionAssays of HeLain situexpressedSPATA2L clone Control Assaywith clone SPATA2L

  25. Experimental Designfor MS assays SDS-PAGE 1D In-situ proteinexpressed Proteinenrichment SDS-PAGE 1D

  26. 1D SDS-Page of in situ expressedproteins Pierce expression of SPATA2L FLJ 82130 CKLPSP2 SB1X WFDC1 GSG1L SB1X SB1X AQP8 SPATA2L C16orf73 MW HSPC 105 ZNFA MW KCNG4 SB1X LOC348174 TMEM170 IRX3 LRRC29 MC1R SB1X MW HAGHL GPR114 C16orf78 DEX1 IG5F6 SB1X

  27. Proteinenrichment of in situ expressedproteins

  28. Electrophoresis of Pierce expression and enrichmentwithMagne-GST beads EnrichmentwithMagne-GST beads of LRRC24. Dil. 1:10 EnrichmentwithMagne-GST beads of SPATA2L SPATA2L by Pierce System. SB1X WB M EnrichmentwithMagne-GST beads of LRRC24. Dil. 1:10 EnrichmentwithMagneyt-GST beads of SPATA2L. Dil. 1:10 MW SB1X SPATA2L by Pierce System. Dil. 1:10

  29. Experimental Designfor MS assays 4 1 2 3 TAG TAG In-situ proteinexpressed Tag IVTT No DNA Empty vector

  30. Others • Bi-lateral ConferenceCalls: CICBiogune-CIC, UCM-CIC,… • WP1 Meeting: Salamanca 29-Nov ( 7 Participants, full-day agenda) • Updated Clones collection & strategyoverview: Agreementwith Director of DNAsu • CommercialAntibodiescollection ( > 6500) at ImmunologyInstitute (Oslo)

  31. Summary • 23/25 proteinsvalidatedbysequencing • 23/25 in situ expressedproteins in Rabbit IVTT • 1/25 in situ expressedproteins in HeLa IVTT • pANT7 compatible withboth IVTT systems • Proteinenrichment • MS assaysin Progress • OtherProteinExpressionStrategiesin Progress • Open collaborationwithDNAsu • Preliminaryplanningfornext 6 months

  32. Acknowledgments CIC Noelia Dasilva Maria Herrero Paula Diez Maria Gonzalez Lucia Lourido Alberto Orfao UCM Concha Gil Felipe Clemente Maria Luisa Elvira Marin CIC Biogune Felix Elorzta Iraide Escobers

  33. Testing Single Proteins in Many Different Ways Perform assays using protein made in bacteria. What other proteins does this protein interact with? Where is the protein located in the cell? Test the protein’s function in mammalian cells. Produce lots of protein for experiments. Turn the protein on and off in mammalian cells.

  34. Gateway Technologyforcloning & subcloning Moving Genes by Recombination Master Only the Desired Product Survives Mix two plasmids and enzyme

  35. InsertVerificationby Xho1 & BamHIDigestion TMEM170 Marcador 1Kb WFDC1 MC1R LRRC29 KCNG4 IGSF6 CKLFSF2 GSG1L

  36. In situ cell-free expression of non-characterizedproteins

  37. Proteínas enviadas

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