Protein Traps

in

AfCS Cells

Dianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser

1. Objective

2. Strategy for WEHI Cells

3. Progress

4. Future Directions

HELPHELP

HELPHELP

HELP

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 DYKDDDDK

FLAG c-mycTEV 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 Cycle

Week 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

westernPassage 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

49

37

26

8363

kDa

TEV protease

FTM Pull-Down

Limitation:

Myc portion of the FTM tag does not work

TAP tag requires both affinity steps

2-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

49

37

26

8363

kDa

TEV protease

FTM Pull-Down

TAP Pull-Down

114

ERK2

RhoB

ERK1-TAP Elution

RhoB-TAP Elution

26

83

63

49

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

49

37

26

8363

kDaScore Score

TEV protease

MS Analysis of SDS/PAGE Gels

1 2 3

4 5 67 8 9

10 11 12

13 14 15

16 17 1819 20

21

YFP Elution

Rho B Elution

Rho C Elution

2223 24

25 26 27

28 29 30

YFP Elution

Rho B Elution

Rho C Elution

Colloidal Coomassie SilverQuest

Co-Eluting Proteins Identified by MS

FTM-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 NA

Enolase 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