protein traps in afcs cells
DESCRIPTION
Protein Traps in AfCS Cells. Dianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser. HELP. HELP. Objective Strategy for WEHI Cells Progress Future Directions. HELP. HELP. HELP. Objectives:. Identify protein-protein interactions in mammalian cells - PowerPoint PPT PresentationTRANSCRIPT
Protein Trapsin
AfCS CellsDianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser
1. Objective
2. Strategy for WEHI Cells
3. Progress
4. Future Directions
HELPHELP
HELPHELPHELP
1. Identify protein-protein interactions in mammalian cells
2. Accomplish this in the AfCS cell line of choice for other studies
Objectives:
1. Design of Bait proteins (Tags)
Requirements
4. Identification of associated proteins by mass spectrometry
3. Isolation of Bait proteins with associated protein complexes
2. Expression of Bait proteins
A Tale of Two Tags
A Strategy for WEHI Cells
FTM Tag for Bait Proteins
N EQKLISEEDL DYKDDDDKFLAG c-myc
TEV protease cleavage site
ENLYFQ CattB
IgG beads
N CBP Protein ATEV protease cleavage site
ENLYFQ Bait CattBCa-CaM
Beads
TAP Tag for Bait Proteins (tandem affinity purification)
Bait
Expression of Bait Protein
1.Transfection
2. Infection with virus
Retroviral Vectors
• Promoter 1: CMV or MSCV, expression level is similar.
• TAG: FTM or TAP tag at the N- or C- terminus.• Selection marker: puromycin or CD4 surface
marker; both work well.
Tag
Promoter 1 Bait gene IRES or promoter2
Selection marker
Progress
A Two Week CycleWeek 1.
Plate packaging cells: eight 100 mm dishes
Mon Tu Wed Thr Fri
Transfection: 8 genes
Harvest virus, store in 4C
Harvest virus, infection
6 million/virus
Remove virus, Passage cells
Passage cells FACS analysis or puromycin select
Week 2. Mon Tu Wed Thr Fri
Sorting by FACS or CD4 beads
western Passage cells(~40 M)
Potential Capacity of 8 viruses/week
• >75 retroviral constructs tested
• ~60 ‘cell lines’ generated xxx(enriched by selection)
• 29 genes
Genes Tested in WEHI-231 Cells
BLNKBtkCalbindinCaMKII deltaCellubrevinClk3Erk1Erk2FurinGrb2
RhoBRhoCRSK3PI3Kp85PI3Kp110Sec22Stat3SykVimentinYFP
Grk2Jnk1LynMAPKK72MEK3Nek4PDE4B3rgRGSRGS4RhoA
= used in pull-downs = poor expression = not yet used in pull-downs
Enrich Expression by FACS
28%before sorting
95%
after sorting
MSCV-FTM-Erk1- Before sorting: ~10 ng/1E6 WEHI cells- After sorting: ~50 ng/1E6 WEHI cells (endogenous Erk1, 25-30 ng/1E6)
FTM-YFP Elution
FTM-ERK2 Elution
ERK2
YFP
114
4937
26
8363
kDa
TEV protease
FTM Pull-Down
Limitation: Myc portion of the FTM tag does not work
TAP tag requires both affinity steps2-Step Pull-Down
ERK2-TAP
IgG beads only – TEV cleavage
CaM beads only – TEV cleavage
TAP-YFP
IgG leaching from the beads
Only nonspecific binding
Purified bait protein
TAP tagged baits can be isolated to essential homogeneity
– but this is not the goal!
Colloidal Coomassie-stained Gels
FTM-YFP Elution
FTM-ERK2 Elution
ERK2
YFP
114
4937
26
8363
kDa
TEV protease
FTM Pull-Down
TAP Pull-Down
114
ERK2
RhoB
ERK1-TAP Elution
RhoB-TAP Elution
26
83
6349
37
kDa
Partial Success with FTM PulldownsExample: FTM-RhoC
• Can reproducibly pull down RhoGDI
• Binds to FTM-RhoC (but not FTM-RhoB)
• Soluble protein, 5-10 g/ml in WEHI lysate
• Represents 0.1-0.2% of total soluble cell protein
• Kd’s for Rho-related proteins are 2-30 nM
MS Analysis of Coll. Coomassie-stained Gel
1 2
3
5 6
784
YFP Elution
ERK2 Elution
ERK2
Identified Proteins
YFP Elution
ERK2 Elution
YFP
1. HSP 90 beta 1x10-11 2. Keratin 1x10-26
3. keratin 1x10-42 4. BSA 2x10-7
7. IgG 2x10-8 8. IgG 6x10-20
5. tropomyosin 5 8x10-12 6. YFP 2x10-33
F-actin capping protein 3x10-7
114
4937
26
8363
kDaScore Score
TEV protease
MS Analysis of SDS/PAGE Gels
1 2 3
4 5 67 8 910 11 12
13 14 15
16 17 1819 20 21
YFP Elution
Rho B Elution
Rho C Elution
22 23 24
25 26 27
28 29 30
YFP Elution
Rho B Elution
Rho C Elution
Colloidal Coomassie SilverQuest
Co-Eluting Proteins Identified by MSFTM-RhoC Score*
RhoGDI 1 6x10-18
14-3-3 theta 1x10-16
Spleen filamen 1x10-12
Tropomodulin 3 1x10-9
Proteosome 28 subunit 2x10-8
RhoGDI 2 3x10-7
Multi-ubiquitin binding protein 4x10-4
Pyruvate kinase 3 1x10-42
Tumor rejection antigen gp96 2x10-14
HSP86 3x10-12
Aldolase 1 5x10-10
Phosphoglycerate kinase 1 2x10-9
Glucose phosphate isomerase 9x10-6
Tropomodulin C 3x10-6
Elongation factor 2 5x10-5
Spleen filamen 3x10-5
HSP 90 beta 1x10-11
tropomyosin 5 8x10-12
F-actin capping protein 3x10-7
Elongation Factor 2 2x10-6
tumor rejection antigen gp96 3x10-5
Kinesin heavy chain NAEnolase 3x10-18
Elongation Factor 1 2 1x10-4
Actin b 9x10-21
MEK3 3x10-29
FTM-RhoB Score
FTM-ERK2 Score FTM-MEK3 Score
* The lower the score, the higher the probability that the protein is correctly identified from the tryptic peptides
Erk2 Associated Proteins in 293 cells • ATP-dependent RNA helicase A• chromatin-specific transcription
elongation factor• splicing Factor 3b, subunit 1 & 2• ribosomal protein S6 kinase
(Rsk2P90)• methylosome protein 50 (MEP50) • Kinesin-related Motor Protein EG5• protein methyltransferase• rErk2• kiaa0122 gene product
• Others…
Erk2
Conclusions regarding FTM-Tagged Baits
• Current protocol can detect a few interacting proteins (RhoGDI, filamen)
• Isolate mostly “nonspecific” proteins
• The abundance of known interacting proteins in WEHI lysate may be too low
1. Design of Bait proteins (Tags)
Summary -- Requirements
4. Identification of associated proteins by mass spectrometry
3. Isolation of Bait proteins with associated protein complexes
2. Expression of Bait proteins OK
OK
Not optimal
Future Directions
• Increased communication with investigators whom have similar paradigms working
• Alternative epitope tags
What’s needed?
1. Better methods: efficient and reproducible
2. New Cells
Solutions
• Choice of cells – (not limited to AfCS cells?)
She ChenShe Chen
Richard DavisRichard Davis
Debalina SiddeeqDebalina Siddeeq
Melissa StalderMelissa Stalder
Joelle ZavzavadjianJoelle Zavzavadjian