Protein Trapsin

AfCS CellsDianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser

1. Objective

2. Strategy for WEHI Cells

3. Progress

4. Future Directions

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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