Working Group 1Mid-term Report

• Action PLAN • Non-characterized protein• Cloning & Validation• Recombinant proteins • Experimental design for MS assays

Strategy Overview

1616 Genes on Chromosome 16

B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast

Entries 538 244 263 491 329

50

150

250

350

450

550

Protein coding genes (n=862)

Entr

ies

More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.

1616 Genes on Chromosome 16

B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast

Entries 538 244 263 491 329

50

150

250

350

450

550

Protein coding genes (n=862)

Entr

ies

More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.

Action Plan

260

CIC collection

25 Non-characterizedproteins

10

Sequence validated & IVTT expressed

August 2012

25

Sequence validated & IVTT expressed

December 2012

Experiments PLATE WELL CLONE ID. VECTOR CLONE NAME

HsxXG003309 H06 HsCD00077640 pANT7_cGST MC1R

HsxXG002436 E04 HsCD00298486 pLDNT7_nFLAG ZNF263

HsxXG003310 A02 HsCD00077481 pANT7_cGST KCNG4

HsxXG003310 B10 HsCD00076975 pANT7_cGST GSG1L

HsxXG006212 B02 HsCD00300882 pANT7_cGST IGSF6

HsxXG006445 F10 HsCD00303939 pANT7_cGST TMEM170

HsxXG003317 E05 HsCD00078574 pANT7_cGST LRRC29

HsxXG003396 C02 HsCD00299942 pANT7_cGST LOC146542

HsxXG006435 D01 HsCD00301997 pANT7_cGST HSPC105

HsxXG006436 F06 HsCD00303761 pANT7_cGST C16orf78

HsxXG006211 F08 HsCD00300816 pANT7_cGST IRX3

HsxXG006211 E12 HsCD00300948 pANT7_cGST CKLFSF2

HsxXG003335 H03 HsCD00077528 pANT7_cGST WFDC1

HsxXG003325 G10 HsCD00076647 pANT7_cGST C16orf73

HsxXG003314 A08 HsCD00077885 pANT7_cGST GPR114

HsxXG006445 F01 HsCD00303878 pANT7_cGST HAGHL

HsxXG003325 E02 HsCD00076537 pANT7_cGST DEXI

HsxXG006449 F02 HsCD00302587 pANT7_cGST SPATA2L

HsxXG006450 D05 HsCD00301981 pANT7_cGST AQP8

HsxXG003400 A06 HsCD00299816 pANT7_cGST FLJ32130

HsxXG006449 A03 HsCD00302589 pANT7_cGST ZNF19

HsxXG006438 A04 HsCD00303645 pANT7_cGST LOC348174

Liquid Culture

O/N, 37ºC, 300 rpm

LB medium + Amp

100 µg/ml+

1.5 µl glycerol stock /clon

DNA Prep

by Pure Yield Plasmid MiniprepPromega system

FastHigh-yield

Semi-automatic

LOC348174

HAGHLZNF19

SPATA2L

GPR114GSG1L

LRRC29 IGSF6

MC1R

KCNG4 CKLFSF2

WFDC1TMEM170

DNA prep

Marcador 1Kb LRRC29 MC1R

KCNG4

TMEM170

GSG1LIGSF6 CKLFSF2

WFDC1

Insert Verification by Xho1 & BamHI Digestion

SEQUENCINGPLATE WELL CLONE ID. VECTOR CLONE NAME SEQUENCE RESULT

HsxXG003309 H06 HsCD00077640 pANT7_cGST MC1RCorrect

HsxXG002436 E04 HsCD00298486 pLDNT7_nFLAG ZNF263 GTPBP6- Sexual Chromosomes

HsxXG003310 A02 HsCD00077481 pANT7_cGST KCNG4 Correct

HsxXG003310 B10 HsCD00076975 pANT7_cGST GSG1L Correct

HsxXG006212 B02 HsCD00300882 pANT7_cGST IGSF6 Correct

HsxXG006445 F10 HsCD00303939 pANT7_cGST TMEM170 Correct

HsxXG003317 E05 HsCD00078574 pANT7_cGST LRRC29 Correct

HsxXG003396 C02 HsCD00299942 pANT7_cGST LOC146542 FLJ32130*HsCD00299816

HsxXG006435 D01 HsCD00301997 pANT7_cGST HSPC105 SDR42E1

HsxXG006436 F06 HsCD00303761 pANT7_cGST C16orf78 Correct

HsxXG006211 F08 HsCD00300816 pANT7_cGST IRX3 Unknown

HsxXG006211 E12 HsCD00300948 pANT7_cGST CKLFSF2 Correct

HsxXG003335 H03 HsCD00077528 pANT7_cGST WFDC1 Correct

HsxXG003325 G10 HsCD00076647 pANT7_cGST C16orf73 Correct

HsxXG003314 A08 HsCD00077885 pANT7_cGST GPR114 Correct

HsxXG006445 F01 HsCD00303878 pANT7_cGST HAGHL Correct

HsxXG003325 E02 HsCD00076537 pANT7_cGST DEXI Correct

HsxXG006449 F02 HsCD00302587 pANT7_cGST SPATA2L Correct

HsxXG006450 D05 HsCD00301981 pANT7_cGST AQP8 Correct

HsxXG003400 A06 HsCD00299816 pANT7_cGST FLJ32130 Correct

HsxXG006449 A03 HsCD00302589 pANT7_cGST ZNF19 Correct

HsxXG006438 A04 HsCD00303645 pANT7_cGST LOC348174 CLEC18A

≥ 90 % sequence validation

•Master Library Many libraries•Any vector can be modified to accept genes

•Master Library Many libraries•Any vector can be modified to accept genes

Recombinant Proteins

Recombinant Proteins

IVTT Others ( E.coli, Pichia, …)

In situ Protein Expression168 µl DEPC water

+ 4 µl Leu or Cys

+ 4 µl Met

+ 16 µl TnT Buffer

+4 µl RNAse out

+ 8 µl T7 polymerase

+0.2-2-0 1.5 µg DNA

90 min, 30ºC, 300 rpm

200 µl Cell lysate

+ PBS

10 min, 4ºC Protein Expressed

In situ cell-free expression of non-characterized proteins

Rabbit Reticulocyte HeLa

TNT coupled Reticulocyte Lysate System

Fast production of Recombinant Protein

Yield per reaction> 0.1-10 µg

23 of 25 non-characterized proteins in situ expressed

IVTT non-coupled from HeLa Lysate System

High-yield In house: >100 µg / reaction

Reagents delivered from Pierce Headquarter ( Madison, WI) on 30th-Nov-2012

1 of 25 non-characterized proteins in situ expressed

1D SDS-Page

MWSB1X SB1X AQP8

LOC348174

SPATA2L

IRX3

C16orf73

TMEM170

FLJ82130

MWSB1X

SB1XGPR114

HSPC105

IG5F6

ZNFA

C16orf78 DEX1

KCNG4

SB1X

SB1XMW

Pierce expression of

SPATA2L

LRRC29

CKLPSP2

MC1R

WFDC1

HAGHL

GSG1L

IVTT Recombinant Proteins

WB Beads Suspension Arrays (BBAs)

WB

IVTT Recombinant Proteins

WB Beads Suspension Arrays (BBAs)

Beads Suspension Assays of HeLa in situ expressed SPATA2L

clone

Control Assay with clone SPATA2L

Beads Suspension Assays of HeLa in situ expressed SPATA2L

clone

Experimental Design for MS assays

In-situ protein expressed

SDS-PAGE 1D

Protein enrichment

SDS-PAGE 1D

1D SDS-Page of in situ expressed proteins

MWSB1X SB1X AQP8

LOC348174

SPATA2L

IRX3

C16orf73

TMEM170

FLJ82130

MWSB1X

SB1XGPR114

HSPC105

IG5F6

ZNFA

C16orf78 DEX1

KCNG4

SB1X

SB1XMW

Pierce expression of

SPATA2L

LRRC29

CKLPSP2

MC1R

WFDC1

HAGHL

GSG1L

Protein enrichment of in situ expressed proteins

SB1X WB M

SPATA2L by Pierce System.

Dil. 1:10

SPATA2L by Pierce System.

Enrichment with Magne-GST beads of LRRC24. Dil. 1:10

Enrichment with Magne-GST beads of LRRC24.

Dil. 1:10

Enrichment with Magne-GST beads of SPATA2L

Enrichment with Magneyt-GST beads of

SPATA2L. Dil. 1:10

MW SB1X

Electrophoresis of Pierce expression and enrichment with

Magne-GST beads

In-situ protein expressed

TAG

TAG

1 2 3 4

Empty vectorTag IVTTNo DNA

Experimental Design for MS assays

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)

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

Acknowledgments

CICNoelia Dasilva Maria Herrero

Paula DiezMaria GonzalezLucia LouridoAlberto Orfao

UCM

Concha Gil Felipe Clemente

Maria LuisaElvira Marin

CIC Biogune

Felix ElorztaIraide Escobers

Testing Single Proteins in Many Different Ways

Where is the protein

located in the cell?

Produce lots of

protein for experiment

s.

Turn the protein on and off in mammalian

cells.

Test the protein’s function in

mammalian cells.

What other proteins does this protein

interact with?

Perform assays using protein made in

bacteria.

Mix two plasmids and enzymeOnly the Desired Product Survives

Master

Moving Genes by Recombination

Gateway Technology for cloning & subcloning

Marcador 1Kb LRRC29 MC1R

KCNG4

TMEM170

GSG1LIGSF6 CKLFSF2

WFDC1

Insert Verification by Xho1 & BamHI Digestion

In situ cell-free expression of non-characterized proteins

Proteínas enviadas