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Protein Traps in AfCS Cells

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

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Protein Traps in AfCS Cells

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  1. Protein Traps in AfCS Cells Dianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser

  2. HELP HELP • Objective • Strategy for WEHI Cells • Progress • Future Directions HELP HELP HELP

  3. Objectives: • Identify protein-protein interactions in mammalian cells • Accomplish this in the AfCS cell line of choice for other studies

  4. Requirements • Design of Bait proteins (Tags) 2. Expression of Bait proteins 3. Isolation of Bait proteins with associated protein complexes • Identification of associated • proteins by mass spectrometry

  5. A Strategy for WEHI Cells A Tale of Two Tags

  6. FTM Tag for Bait Proteins TEV protease cleavage site c-myc FLAG N DYKDDDDK ENLYFQ EQKLISEEDL attB Bait C TAP Tag for Bait Proteins (tandem affinity purification) N Protein A ENLYFQ CBP attB Bait C Ca-CaM Y TEV protease cleavage site IgG beads Beads

  7. Expression of Bait Protein • Transfection • 2. Infection with virus

  8. IRES or promoter2 Promoter 1 Bait gene Selection marker Retroviral Vectors Tag • 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.

  9. Progress

  10. A Two Week Cycle Week 1. Mon Tu Wed Thr Fri Transfection: 8 genes Harvest virus, store in 4C Harvest virus, infection 6 million/virus Remove virus, Passage cells Plate packaging cells: eight 100 mm dishes Week 2. Tu Mon Wed Thr Fri Passage cells FACS analysis or puromycin select Sorting by FACS or CD4 beads western Passage cells (~40 M) Potential Capacity of 8 viruses/week

  11. >75 retroviral constructs • tested • ~60 ‘cell lines’ generated xxx(enriched by selection) • 29 genes

  12. BLNK Btk Calbindin CaMKII delta Cellubrevin Clk3 Erk1 Erk2 Furin Grb2 Genes Tested in WEHI-231 Cells = used in pull-downs = poor expression = not yet used in pull-downs Grk2 Jnk1 Lyn MAPKK7a2 MEK3 Nek4 PDE4B3 rgRGS RGS4 RhoA • RhoB • RhoC • RSK3 • PI3Kp85b • PI3Kp110a • Sec22 • Stat3 • Syk • Vimentin • YFP

  13. after sorting 95% before sorting 28% Enrich Expression by FACS MSCV-FTM-Erk1 - Before sorting: ~10 ng/1E6 WEHI cells - After sorting: ~50 ng/1E6 WEHI cells (endogenous Erk1, 25-30 ng/1E6)

  14. FTM Pull-Down FTM-ERK2 Elution FTM-YFP Elution kDa Limitation: Myc portion of the FTM tag does not work 114 83 63 ERK2 49 37 YFP 26 TEV protease

  15. TAP tag requires both affinity steps IgG beads only – TEV cleavage CaM beads only – TEV cleavage 2-Step Pull-Down ERK2-TAP TAP-YFP IgG leaching from the beads Purified bait protein Only nonspecific binding

  16. TAP tagged baits can be isolated to essential homogeneity – but this is not the goal!

  17. Colloidal Coomassie-stained Gels FTM Pull-Down TAP Pull-Down FTM-ERK2 Elution FTM-YFP Elution RhoB-TAP Elution ERK1-TAP Elution kDa kDa 114 114 83 63 83 ERK2 49 63 ERK2 37 YFP 49 26 37 TEV protease RhoB 26

  18. Partial Success with FTM PulldownsExample: FTM-RhoC • Can reproducibly pull down RhoGDI • Binds to FTM-RhoC (but not FTM-RhoB) • Soluble protein, 5-10 mg/ml in WEHI lysate • Represents 0.1-0.2% of total soluble cell protein • Kd’s for Rho-related proteins are 2-30 nM

  19. MS Analysis of Coll. Coomassie-stained Gel ERK2 Elution YFP Elution Identified Proteins ERK2 Elution YFP Elution kDa Score Score 114 1. HSP 90 beta 1x10-112.Keratin 1x10-26 1 2 83 3. keratin 1x10-42 4. BSA 2x10-7 4 3 8 63 7. IgG 2x10-8 8. IgG 6x10-20 7 ERK2 49 37 5. tropomyosin 58x10-12 6. YFP 2x10-33 F-actin capping protein 3x10-7 YFP 6 5 26 TEV protease

  20. MS Analysis of SDS/PAGE Gels Colloidal Coomassie SilverQuest 1 2 3 22 24 23 25 26 27 4 5 6 7 9 8 10 11 12 14 15 13 28 29 30 17 16 18 20 19 21 YFP Elution YFP Elution Rho C Elution Rho B Elution Rho C Elution Rho B Elution

  21. 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

  22. 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

  23. Summary -- Requirements Not optimal • Design of Bait proteins (Tags) OK 2. Expression of Bait proteins 3. Isolation of Bait proteins with associated protein complexes • Identification of associated • proteins by mass spectrometry OK

  24. Future Directions What’s needed? Solutions • Better methods: efficient and reproducible • Increased communication with investigators whom have similar paradigms working • Alternative epitope tags 2. New Cells • Choice of cells – (not limited to AfCS cells?)

  25. She Chen Richard Davis Debalina Siddeeq Melissa Stalder Joelle Zavzavadjian

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