Protein Trapsin
AfCS CellsDianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser
1. Objective
2. Strategy for WEHI Cells
3. Progress
4. Future Directions
HELPHELP
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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