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Working Group 1 Mid-term Report
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Strategy Overview Action PLAN Non-characterized protein
Cloning & Validation Recombinant proteins Experimental design for MS assays
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16 Genes on Chromosome 16 More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.
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16 Genes on Chromosome 16 More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.
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Action Plan 260 CIC collection 25 Non-characterized proteins 25 10
December 2012 10 August 2012 Sequence validated & IVTT expressed Sequence validated & IVTT expressed
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Experiments PLATE WELL CLONE ID. VECTOR CLONE NAME HsxXG003309 H06
HsCD pANT7_cGST MC1R HsxXG002436 E04 HsCD pLDNT7_nFLAG ZNF263 HsxXG003310 A02 HsCD KCNG4 B10 HsCD GSG1L HsxXG006212 B02 HsCD IGSF6 HsxXG006445 F10 HsCD TMEM170 HsxXG003317 E05 HsCD LRRC29 HsxXG003396 C02 HsCD LOC146542 HsxXG006435 D01 HsCD HSPC105 HsxXG006436 F06 HsCD C16orf78 HsxXG006211 F08 HsCD IRX3 E12 HsCD CKLFSF2 HsxXG003335 H03 HsCD WFDC1 HsxXG003325 G10 HsCD C16orf73 HsxXG003314 A08 HsCD GPR114 F01 HsCD HAGHL E02 HsCD DEXI HsxXG006449 F02 HsCD SPATA2L HsxXG006450 D05 HsCD AQP8 HsxXG003400 A06 HsCD FLJ32130 A03 HsCD ZNF19 HsxXG006438 A04 HsCD LOC348174
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1.5 µl glycerol stock /clon
Liquid Culture LB medium + Amp 100 µg/ml + 1.5 µl glycerol stock /clon O/N, 37ºC, 300 rpm
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by Pure Yield Plasmid MiniprepPromega system
DNA Prep by Pure Yield Plasmid MiniprepPromega system Fast High-yield Semi-automatic
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DNA prep MC1R TMEM170 WFDC1 LOC348174 ZNF19 SPATA2L HAGHL LRRC29 IGSF6
GSG1L KCNG4 CKLFSF2 GPR114
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Insert Verification by Xho1 & BamHI Digestion
Marcador 1Kb LRRC29 MC1R KCNG4 TMEM170 GSG1L IGSF6 CKLFSF2 WFDC1
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GTPBP6- Sexual Chromosomes
SEQUENCING PLATE WELL CLONE ID. VECTOR CLONE NAME SEQUENCE RESULT HsxXG003309 H06 HsCD pANT7_cGST MC1R Correct HsxXG002436 E04 HsCD pLDNT7_nFLAG ZNF263 GTPBP6- Sexual Chromosomes HsxXG003310 A02 HsCD KCNG4 B10 HsCD GSG1L HsxXG006212 B02 HsCD IGSF6 HsxXG006445 F10 HsCD TMEM170 HsxXG003317 E05 HsCD LRRC29 HsxXG003396 C02 HsCD LOC146542 FLJ32130*HsCD HsxXG006435 D01 HsCD HSPC105 SDR42E1 HsxXG006436 F06 HsCD C16orf78 HsxXG006211 F08 HsCD IRX3 Unknown E12 HsCD CKLFSF2 HsxXG003335 H03 HsCD WFDC1 HsxXG003325 G10 HsCD C16orf73 HsxXG003314 A08 HsCD GPR114 F01 HsCD HAGHL E02 HsCD DEXI HsxXG006449 F02 HsCD SPATA2L HsxXG006450 D05 HsCD AQP8 HsxXG003400 A06 HsCD FLJ32130 A03 HsCD ZNF19 HsxXG006438 A04 HsCD LOC348174 CLEC18A ≥ 90 % sequence validation
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Recombinant Proteins Master Library Many libraries
Any vector can be modified to accept genes
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Recombinant Proteins IVTT Others ( E.coli, Pichia, …)
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In situ Protein Expression
168 µl DEPC water + 4 µl Leu or Cys 4 µl Met 16 µl TnT Buffer 4 µl RNAse out 8 µl T7 polymerase µg DNA + PBS 200 µl Cell lysate 90 min, 30ºC, 300 rpm 10 min, 4ºC Protein Expressed
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In situ cell-free expression of non-characterized proteins
Rabbit Reticulocyte HeLa
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TNT coupled Reticulocyte Lysate System
Fast production of Recombinant Protein Yield per reaction > µg 23 of 25 non-characterized proteins in situ expressed
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IVTT non-coupled from HeLa Lysate System
High-yield In house: >100 µg / reaction
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Reagents delivered from Pierce Headquarter ( Madison, WI) on 30th-Nov-2012
1 of 25 non-characterized proteins in situ expressed
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Pierce expression of SPATA2L
1D SDS-Page MW SB1X AQP8 LOC348174 SPATA2L IRX3 C16orf73 TMEM170 FLJ 82130 GPR114 HSPC 105 IG5F6 ZNFA C16orf78 DEX1 KCNG4 Pierce expression of SPATA2L LRRC29 CKLPSP2 MC1R WFDC1 HAGHL GSG1L
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IVTT Recombinant Proteins
WB Beads Suspension Arrays (BBAs)
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WB
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IVTT Recombinant Proteins
WB Beads Suspension Arrays (BBAs)
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Beads Suspension Assays of HeLa in situ expressed SPATA2L clone
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Beads Suspension Assays of HeLa in situ expressed SPATA2L clone
Control Assay with clone SPATA2L
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Experimental Design for MS assays
SDS-PAGE 1D In-situ protein expressed Protein enrichment SDS-PAGE 1D
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1D SDS-Page of in situ expressed proteins
MW SB1X AQP8 LOC348174 SPATA2L IRX3 C16orf73 TMEM170 FLJ 82130 GPR114 HSPC 105 IG5F6 ZNFA C16orf78 DEX1 KCNG4 Pierce expression of SPATA2L LRRC29 CKLPSP2 MC1R WFDC1 HAGHL GSG1L
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Protein enrichment of in situ expressed proteins
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Electrophoresis of Pierce expression and enrichment with Magne-GST beads
Enrichment with Magne-GST beads of LRRC24. Dil. 1:10 Enrichment with Magne-GST beads of SPATA2L SB1X WB M SPATA2L by Pierce System. SPATA2L by Pierce System. Dil. 1:10 Enrichment with Magne-GST beads of LRRC24. Dil. 1:10 Enrichment with Magneyt-GST beads of SPATA2L. Dil. 1:10 MW SB1X
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Experimental Design for MS assays
4 1 2 3 TAG TAG In-situ protein expressed Tag IVTT No DNA Empty vector
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Others Bi-lateral Conference Calls: CICBiogune-CIC, UCM-CIC,…
WP1 Meeting: Salamanca 29-Nov ( 7 Participants, full-day agenda) Updated Clones collection & strategy overview: Agreement with Director of DNAsu Commercial Antibodies collection ( > 6500) at Immunology Institute (Oslo)
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Summary 23/25 proteins validated by sequencing
23/25 in situ expressed proteins in Rabbit IVTT 1/25 in situ expressed proteins in HeLa IVTT pANT7 compatible with both IVTT systems Protein enrichment MS assays in Progress Other Protein Expression Strategies in Progress Open collaboration with DNAsu Preliminary planning for next 6 months
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Acknowledgments CIC UCM CIC Biogune 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
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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.
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Gateway Technology for cloning & subcloning
Moving Genes by Recombination Master Only the Desired Product Survives Mix two plasmids and enzyme
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Insert Verification by Xho1 & BamHI Digestion
Marcador 1Kb LRRC29 MC1R KCNG4 TMEM170 GSG1L IGSF6 CKLFSF2 WFDC1
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In situ cell-free expression of non-characterized proteins
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Proteínas enviadas
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