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May 2017
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An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
靶向破坏内源蛋白的亲和力定向蛋白导弹(AdPROM)系统   

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Abstract

We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et al., 2014; Schmidt et al., 2016). In this protocol, we describe detailed methodology involved in generating AdPROM constructs and their application in human cell lines for target protein destruction. AdPROM allows functional characterisation of the POI and its efficiency of target protein destruction overcomes many limitations of RNA-interference approaches, which necessitate long treatments and are associated with off-target effects, and CRISPR/Cas9 gene editing, which is not always feasible.

Keywords: AdPROM (AdPROM), Proteolysis (蛋白水解), VHL (VHL), Cullin2 (Cullin2), Ubiquitination (泛素化), Nanobody (纳米抗体), Monobody (单抗体), CRISPR/Cas9 (CRISPR/Cas9)

Background

This protocol enables one to design, build and express AdPROM VHL-nano/monobody constructs in mammalian cell lines to achieve the proteolytic destruction of the endogenous POI. In the original entries, we demonstrated the near-complete destruction of specific target proteins, by using nanobodies that recognise either green fluorescent protein (GFP) (Fridy et al., 2014) or the inflammasomal protein ASC (Schmidt et al., 2016) and two distinct monobodies that recognize the protein tyrosine phosphatase SHP2 (Sha et al., 2013) as target probes, in a number of human cancer cell lines (Fulcher et al., 2016 and 2017). This protocol provides details for the generation of AdPROM constructs, their expression in cells, and monitoring of target protein degradation and can be adapted for use with any nanobody and monobody, both for constitutive and inducible degradation of the POI. The focus of this protocol is not on the generation of nano/monobodies against POIs or CRISPR/Cas9 genome editing (to knockin GFP tags on POIs) but rather the latter steps to facilitate target protein destruction with the AdPROM system.

Materials and Reagents

  1. Pipette tips (10 µl, 200 µl, 1,000 µl, alpha gel loading tips) (Greiner Bio One International, catalog numbers: 771290; STARLAB INTERNATIONAL, catalog number: S1111-1006 ; Greiner Bio One International, catalog number: 740295 and Alpha Laboratories, catalog number: LW1100 respectively)
  2. 15 ml Falcons tube (Greiner Bio One International, catalog number: 188271 )
  3. 50 ml Falcon tube (Greiner Bio One International, catalog number: 227261 )
  4. 10-cm tissue culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 172931 )
  5. Micro tubes (1.5 ml) (SARSTEDT, catalog number: 72.706.400 )
  6. 0.45 µm sterile syringe filters (Sartorius, catalog number: 16555-K )
  7. 0.22 µm sterile syringe filters (Sartorius, catalog number: 16532-K )
  8. 96-well plates for tissue culture (Greiner Bio One International, catalog number: 655101 )
  9. Immobilon-®P PVDF membranes (Merck, catalog number: IPFL00005 )
  10. 10 ml plastic syringes (BD, BD Biosciences, catalog number: 302188 )
  11. Sterile disposable scalpels (Swann Morton, catalog number: 0503 )
  12. X-ray films (Konica Minolta, APLUS)
  13. HEK-293 FT cells for retrovirus production (Thermo Fisher Scientific, InvitrogenTM, catalog number: R70007 )
  14. Cell line of interest that expresses the target protein
    Note: We used U2OS osteosarcoma, Human Embryonic kidney HEK-293, adenocarcinoma A549, and breast cancer MDA-MB-231 and MDA-MB-468 cells in the original entries (Fulcher et al., 2016 and 2017).
  15. Cloning grade chemically competent DH5α cells (prepared in-house using a modified version of the Hanahan method (Sambrook and Russell, 2006))
  16. pBABED (Dundee modified pBABE vector) Puro FLAG vectors containing controls and AdPROM reagents for retrovirus production
    Note: These may be obtained from the MRCPPU http://mrcppureagents.dundee.ac.uk/reagent-catalogues (refer to AdPROM cloning procedure below for details).
  17. pCMV-GAG/Pol (Cell Biolabs, catalog number: RV-111 )
  18. pCMV-VSVG (Cell Biolabs, catalog number: RV-110 )
  19. pRetroX-Tet-On Advanced system plasmids for Tet-inducible AdPROM expression (Takara Bio, Clontech, catalog number: 632104 )
  20. Nano/monobody cDNA with flanking EcoRI/NotI sites (the sequences for the nano/monobodies constructs used in the original entries were obtained from the literature (Fulcher et al., 2016 and 2017))
  21. Restriction enzymes (FastDigest)–BamHI, EcoRI, DpnI and NotI (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: FD0054 , FD0274 , FD1703 and FD0594 respectively)
  22. Ultrapure agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  23. Sequencing oligos (0.025 nM, Desalted) (Sigma-Aldrich)
  24. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  25. Rapid DNA Ligation Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1422 )
  26. Ampicillin (ForMedium, catalog number: AMP25 )
  27. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
  28. PureLinkTM HiPure Plasmid Filter Maxiprep Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K210017 )
  29. KOD Hot Start DNA polymerase (Merck, catalog number: 71086-3 )
  30. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  31. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  32. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11960085 )
  33. Foetal bovine serum (FBS) (Labtech, catalog number: FCS-SA/500 )
  34. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
  35. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  36. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  37. Cell culture grade trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  38. Polyethylenimine (PEI) (Polysciences, catalog number: 24765 )
  39. HEPES (Sigma-Aldrich, catalog number: H4034 )
  40. Polybrene (Sigma-Aldrich, catalog number: 107689 )
  41. Puromycin (Sigma-Aldrich, catalog number: P9620 )
  42. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190169 )
  43. Non-fat dried milk powder (we use Marvel milk powder)
  44. 4-12% Bis/Tris gradient gels (Novex)
  45. Primary antibody that recognises the POI
    Note: We used in-house generated antibodies recognizing VPS34 and FAM83G in the first of the original entries (Fulcher et al., 2016). Both antibodies can be made available upon request or purchased from the MRC-PPU Reagents Website (http://mrcppureagents.dundee.ac.uk/reagent-catalogues). For the second original entry (Fulcher et al., 2017), we used anti-SHP2 (C-terminus (Cell Signaling Technology, catalog number: 3397 ); and N-terminus (Cell Signaling Technology, catalog number: 3752 )) and anti-ASC (Martin Oeggerli, Adipogen, catalog number: AL177 ) antibodies.
  46. Primary antibodies that recognise GFP (an anti-GFP antibody from ChromoTek, catalog number: 3H9), and in-house generated anti-GFP antibody, which can be purchased from the MRC-PPU Reagents Website (http://mrcppureagents.dundee.ac.uk/reagent-catalogues)
  47. Primary antibody that recognises VHL (Cell Signalling Technology, catalog number: 68547 )
  48. Primary antibody that recognises a house keeping gene (loading control) (We use anti-GAPDH (Cell Signaling Technology, catalog number: 2118 ))
  49. Bovine serum albumin (BSA) powder (Sigma-Aldrich, catalog number: A7906 )
  50. Enhanced Chemiluminescence (ECL) reagent (GE Healthcare, catalog number: RPN2106 )
  51. Secondary antibodies for primary antibody identification
    Note: We use anti-sheep IgG-HRP (Santa Cruz Biotechnology, catalog number: sc-2770 ); and anti-rabbit IgG, HRP-linked (Cell Signaling Technology, catalog number: 7074 )
  52. G418/Geneticin (Thermo Fisher Scientific, GibcoTM, catalog number: 10131035 )
  53. Doxycycline (hydrochloride) (Sigma-Aldrich, catalog number: D3447 )
  54. Orange G (Sigma-Aldrich, catalog number: O3756 )
  55. Glycerol (VWR, catalog number: 24388.320 )
  56. Ethylenediaminetetraacetic acid (EDTA) (ForMedium, catalog number: EDTA250 )
  57. Tris (VWR, catalog number: 103157P )
  58. Sucrose (VWR, catalog number: 27480.360 )
  59. Sodium chloride (NaCl) (VWR, catalog number: 27810.364 )
  60. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  61. Sodium orthovanadate (Sigma-Aldrich, catalog number: 450243 )
  62. β-Glycerophosphate (Sigma-Aldrich, catalog number: G9422 )
  63. Sodium fluoride (Sigma-Aldrich, catalog number: S7920 )
  64. Sodium pyrophosphate (Sigma-Aldrich, catalog number: P8010 )
  65. Nonidet P-40 substitute (Sigma-Aldrich, catalog number: 74385 )
  66. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  67. Protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 11836170001 )
  68. Glycine (VWR, catalog number: 10119CU )
  69. Methanol (VWR, catalog number: 20847.307 )
  70. Sodium dodecyl sulphate (SDS) (VWR, catalog number: 444464T )
  71. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  72. Hydrochloric acid (HCl) for pH adjustment (VWR, catalog number: 20252.335 )
  73. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  74. Gelatin (from porcine skin) (Sigma-Aldrich, catalog number: G2500 )
  75. DNA loading buffer (see Recipes)
  76. Lysis buffer (see Recipes)
  77. Running buffer (10x) (see Recipes)
  78. Transfer buffer (10x) (see Recipes)
  79. Sample buffer (5x) (see Recipes)
  80. TBS (10x) (see Recipes)
  81. TBS-T (1x) (see Recipes)
  82. TE buffer (pH 8.0) (see Recipes)

Equipment

  1. Water baths 37 °C and 42 °C
  2. Incubator/shaker 37 °C (Infors)
  3. Desktop centrifuge (4 °C)
  4. Desktop centrifuge (RT [room temperature])
  5. 500 ml Erlenmeyer flask
  6. Thermocycler (Thermo Fisher Scientific, Applied Biosystems, model: ProFLEX PCR System )
  7. Vortex (Scientific Industries, model: Vortex-Genie 2 )
  8. Humidified incubator for cell culture
  9. Sterile hood for tissue culture, suitable for category 2 work
  10. Pipettes (P2, P20, P200, P1000)
  11. Table-top heat block
  12. Gel electrophoresis apparatus (Peqlab)
  13. X-ray film developer
  14. Autoclave
  15. Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 )

Procedure

  1. Generation of AdPROM constructs for constitutive expression in cells
    1. Preparation of inserts
      1. Synthesise the specific nano/monobody binding domain using GeneArt (Life Technologies) with flanking 5’-EcoRI and 3’-NotI sites as follows: GAATTCGCCATG-(nano/mono)-TGAGCGGCCGC. If the sequence contains internal EcoRI or NotI sites then introduce silent changes to remove them such that the flanking sites are unique, it may also be useful to codon optimise according to the cell line to be targeted.
      2. Re-suspend the lyophilised GeneArt vector in 50 µl d/dH2O (distilled/deionised H2O).
      3. Digest 25 µl of the re-suspended GeneArt vector with EcoRI and NotI at 37 °C for 2 h; (25 µl DNA, 10 µl FastDigest Green Buffer, 2 µl EcoRI, 2 µl NotI, 61 µl d/dH2O).
      4. Run the entire digest on a 1% agarose gel (130 V, 35 min) (split between neighbouring wells as necessary). Two bands should be visible; the heavier vector band should run at approximately 2.3-2.5 Kb depending on the GeneArt backbone provided and the released insert band should run at the expected size of the synthesized nano/monobody binding domain (see Figure 1 for a simulated DNA digest gel image). Excise the released cDNA insert (small band). For DNA loading buffer (see Recipes).
      5. Purify the digested insert using the QIAquick gel extraction kit according to the manufacturer’s instructions and elute in 50 µl d/dH2O.


        Figure 1. Simulated DNA agarose gel highlighting the expected products obtained in each digest. Molecular weights (MW) are in base pairs (bp). 1. (Step A1c) pMK-RQ AdPROM (GeneArt synthesized) x EcoRI + NotI (upper band = vector, 2,286 bp, lower band = insert, 307 bp). 2. (Step A2b) pBABED P FLAG VHL 5xGly no stop (DU54795) x EcoRI + NotI, 5,830 bp. 3. (Step A2b) pBABED P FLAG (DU37983) x EcoRI + NotI, 5,176 bp. 4. (Step B2a) pRetroX-Tight Puro x EcoRI + NotI, 6,536 bp. 5. (Step B2i; Step B3j) pRetroX-Tight Puro x BamHI + NotI, 6,560 bp. 6. (Step B3c) VHL PCR x BamHI + NotI, 664 bp. 7. (Step B2e) Nano/monobody PCR x BamHI + NotI, 307 bp.

    2. Preparation of vectors
      1. Digest accepting vectors: for the AdPROM construct, cut pBABED Puro FLAG VHL (DU54795) and for the nano/monobody only control cut pBABED Puro FLAG (DU37983). An additional VHL only control vector is available if required (DU54477). All constitutive expression AdPROM reagents and vectors may be ordered online from the MRC-PPU (http://mrcppureagents.dundee.ac.uk/reagent-catalogues).
      2. Digest each accepting vector with EcoRI and NotI at 37 °C for 2 h; (3-5 µg vector, 10 µl FastDigest Green Buffer, 2 µl EcoRI, 2 µl NotI, d/dH2O to 100 µl final volume). pBABED Puro FLAG VHL (DU54795) and pBABED Puro FLAG (DU37983) should produce single fragments of 5.9 Kb and 5.2 Kb respectively (Figure 1).
      3. Run the entire digest on a 1% agarose gel (130 V, 35 min) (split between neighbouring wells as necessary) and excise the cut vector band (single large band).
      4. Purify the digested vectors using the QIAquick gel extraction kit and elute in 50 µl d/dH2O.
    3. Cloning, verification and Maxiprep
      1. Ligate the AdPROM insert into both the empty and VHL-containing accepting vectors using the Rapid DNA Ligation Kit (5 µl insert, 1 µl vector, 4 µl 5x Rapid DNA Ligation Buffer, 1 µl Ligase, 9 µl d/dH2O).
      2. Mix well and incubate for 5 min at RT.
      3. Add 5 µl of the ligation reaction to 50 µl competent DH5α cells on ice and leave for 2 min.
      4. Move cells to 42 °C water bath for 45 sec to heat shock then place on ice for 1 min.
      5. Plate the entire contents of the transformation tube onto an LB (lysogeny broth) agar plate containing 100 µg/ml ampicillin and place O/N (overnight) at 37 °C.
        Note: Ampicillin is a slow-acting antibiotic thus there is no need for a recovery step before plating.
      6. The following morning, pick 4-6 colonies from the plate and add each to 4 ml LB (50 µg/ml ampicillin) in 15 ml Falcons. Propagate O/N in a shaker incubator at 37 °C, 220 rpm.
      7. After 16 h growth, pellet cells by centrifugation at 2,000 x g for 10 min at 4 °C.
      8. Harvest plasmid DNA from each pellet using the QIAprep Spin Miniprep kit according to the manufacturer’s instructions and elute in 50 µl d/dH2O.
      9. Set up a small 20 µl diagnostic digest for each and incubate for 1 h at 37 °C; (6 µl Plasmid DNA, 2 µl FastDigest Green Buffer, 0.1 µl EcoRI, 0.1 µl NotI, 11.8 µl d/dH2O).
      10. Run the digested samples on a 1% agarose gel and look for the excision of the AdPROM insert. Two bands should be visible; the heavier vector band should run at approximately 4.3 Kb and the smaller released insert band should run at the expected size of the synthesized nano/monobody binding domain.
      11. Sequence 1 or 2 positives using primers pBABE F (5’-CCTCCTCTTCCTCCATCC) and pBABE R (5’-CCACACCTGGTTGCTGACTAATTGAG).
      12. Transform 50 µl DH5α cells with 5 ng of plasmid DNA from the confirmed clone and plate on an LB agar plate (100 µg/ml ampicillin).
      13. The following morning pick a single colony and inoculate 10 ml LB (50 µg/ml ampicillin) in a 50 ml Falcon tube and grow for 6-7 h at 37 °C, 220 rpm.
      14. Add the entire contents to a 500 ml Erlenmeyer flask containing 150 ml LB (50 µg/ml ampicillin) and grow overnight at 37 °C, 220 rpm.
      15. Harvest plasmid from the culture using the PureLinkTM HiPure Plasmid Filter Maxiprep Kit according to the manufacturer’s instructions and elute the purified plasmid DNA in 1 ml TE buffer (pH 8.0).
      16. Measure the DNA concentration using a NanoDrop instrument in preparation for downstream transfection procedures; and verify the Maxi-prepped vector(s) by both restriction digestion and DNA sequencing.

  2. Generation of AdPROM constructs for Tetracycline (Tet)-inducible expression in cells
    1. Nanobody generation and primer design
      1. Synthesise the specific nano/monobody using GeneArt (Life Technologies) with flanking 5’-EcoRI and 3’-NotI sites as follows: GAATTCGCCATG-(nano/monobody)-TGAGCGGCCGC. If the sequence contains internal BamHI, EcoRI or NotI sites then introduce silent changes to remove them such that the flanking sites are unique, it may also be useful to codon optimise according to the cell-line to be targeted.
      2. The multiple cloning site of the pRetroX-Tight Puro destination vector is limited, thus it is necessary to order additional primers to PCR amplify the AdPROM insert with flanking BamHI and NotI sites: gctaGGATCCGCCATG-(nano/monobody)-TGAGCGGCCGCgact (the lower case bases indicate overhangs to allow direct digestion of PCR product). A Tm of 60 °C is recommended for PCR primers; if lower then alter the annealing temperature of any reactions accordingly.
      3. Design oligonucleotides to amplify VHL (variant 1, NM_000551.3) with a 3’-5xGLY linker, no stop codon and flanking 5’-BamHI and 3’-EcoRI restriction sites; gctaGGATCCGCC-(VHL)-GGTGGAGGCGGAGGTGAATTCgact (the lower case bases indicate overhangs to allow direct digestion of the PCR product). A holding vector (DU52906) containing this cloned fragment is available from the MRC-PPU (http://mrcppureagents.dundee.ac.uk/reagent-catalogues).
    2. Generation of Tet-inducible nano/monobody control
      1. Digest the vector pRetroX-Tight Puro with BamHI and NotI to produce a single fragment of 6.6 Kb (Figure 1), purifying as shown above (step A2) and eluting in 50 µl d/dH2O.
      2. Re-suspend the lyophilised GeneArt nano/monobody vector in 50 µl d/dH2O.
      3. PCR amplify the nano/monobody with BamHI and NotI primers using KOD Hot Start DNA polymerase (1 µl template [10 ng/µl GeneArt cDNA], 5 µl 10x PCR buffer, 5 µl dNTP mix [2 mM each], 3 µl 25 mM MgSO4, 3 µl DMSO, 1.5 µl each F&R primers [10 µM], 1 µl KOD polymerase, 29 µl d/dH2O).
      4. PCR: 2 min 94 °C then 30 cycles of 15 sec 94 °C, 30 sec 60 °C, and 25 sec 72 °C followed by a single elongation step of 7 min 72 °C.
      5. Run 5 µl of the PCR reaction with 15 µl loading buffer on a 1% agarose gel to determine success. The PCR band should match the size of the nano/monobody template (Figure 1).
      6. Clean 40 µl of the confirmed PCR reaction (step B2d) using the QIAquick PCR purification kit according to the manufacturer’s instructions but eluting in 85 µl d/dH2O.
      7. Cut the entire cleaned PCR product by adding 10 µl 10x FastDigest buffer, 2 µl BamHI, 2 µl NotI and 1 µl DpnI to the eluate from step B2f. Incubate at 37 °C for a minimum of 2 h.
      8. Repeat the PCR cleanup step, this time eluting in 50 µl d/dH2O to yield the digested AdPROM insert.
      9. Ligate the AdPROM insert into pRetroX-Tight Puro following the cloning, verification and Maxiprep steps shown previously (section A3) except use BamHI and NotI in step A3i (Figure 1), sequence with pBABE F (5’-CCTCCTCTTCCTCCATCC) and pRetroX R (5’-TGCTCCAGACTGCCTTGG) in step A3k and inoculate 250-400 ml of LB (50 µg/ml ampicillin) in step A3n.
    3. Generation of Tet-inducible AdPROM vector
      1. Set up a VHL PCR using KOD Hot Start DNA polymerase; (1 µl template [VHL containing vector, section A3 above]), 5 µl 10x PCR buffer, 5 µl dNTP mix (2 mM each), 3 µl 25 mM MgSO4, 3 µl DMSO, 1.5 µl each F&R primers (10 µM), 1 µl KOD polymerase, 29 µl d/dH2O.
      2. PCR: 2 min 94 °C then 30 cycles of 15 sec 94 °C, 30 sec 60 °C, and 50 sec 72 °C followed by a single elongation step of 7 min 72 °C.
      3. Run 5 µl of the PCR reaction with 15 µl loading buffer on a 1% agarose gel to determine success. The expected band size for VHL and linker should be 0.7 Kb (Figure 1).
      4. Clean 40 µl of the confirmed PCR reaction (step B3b) using QIAGEN’s QIAquick PCR purification kit according to manufacturer’s instructions but eluting in 85 µl d/dH2O.
      5. Cut the entire cleaned PCR product by adding 10 µl 10x FastDigest buffer, 2 µl BamHI, 2 µl EcoRI and 1 µl DpnI to the eluate from step B3d. Incubate at 37 °C for a minimum of 2 h then repeat the PCR cleanup step, this time eluting in 50 µl d/dH2O to yield the digested VHL insert.
      6. Digest 25 µl of the re-suspended GeneArt nano/monobody vector with EcoRI and NotI at 37 °C for 2 h (25 µl DNA, 10 µl 10x FastDigest Green Buffer, 2 µl EcoRI, 2 µl NotI, 61 µl d/dH2O).
      7. Run the entire digest on a 1% agarose gel (split between neighbouring wells as necessary) and excise the released cDNA insert (small band).
      8. Purify using the QIAquick gel extraction kit and elute in 50 µl d/dH2O to yield the AdPROM insert.
      9. Perform a 3-way ligation (see Figure 2) by simultaneously inserting the VHL and AdPROM inserts into pRetroX-Tight Puro using the Rapid DNA Ligation Kit; (2.5 µl VHL insert (BamHI/EcoRI), 2.5 µl AdPROM insert (EcoRI/NotI), 1 µl pRetroX-Tight Puro vector (BamHI/NotI), 4 µl 5x Rapid DNA Ligation Buffer, 1 µl ligase, 9 µl d/dH2O).
      10. Follow the cloning, verification and Maxiprep steps shown previously (section A3) with some minor changes; allow 10 min for the ligation reaction in Procedure A3b, add 10 µl of ligation reaction to 100 µl cells in step A3c, perform diagnostic digests in Procedure E using BamHI and NotI (Figure 1), sequence with pBABE F (5’-CCTCCTCTTCCTCCATCC) and pRetroX R (5’-TGCTCCAGACTGCCTTGG) in step A3k and inoculate 250-400 ml of LB (50 µg/ml ampicillin) in step A3m.


        Figure 2. Schematic illustrating a typical 3-way ligation as described in step B3i. The diagram shows the Tet-inducible pRetroX Tight plasmid (see Procedure E), however, the principles for pBABED plasmids remain the same.

  3. Retrovirus production and target cell infection for the constitutive expression AdPROM system
    1. Culture HEK-293 FT cells and the desired target cell lines following the recommended conditions. For HEK-293 FT cells, cells are grown in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin (hereafter referred to as complete medium). Cells are maintained at 37 °C in a humidified incubator at 5% CO2.
    2. Seed 5 x 105 HEK-293 FT cells per one 10-cm culture dish in complete medium and incubate at 37 °C in a humidified incubator at 5% CO2 overnight. The next day the cells should be ~70% confluent. Prior to transfection, replace medium with 9 ml of complete medium. One 10-cm dish of ~70% confluent HEK-293 FT cells is enough for the generation of an individual retrovirus. For the constitutive expression AdPROM system we recommend the following control and AdPROM retroviruses: Flag-tagged Nano/monobody alone, Flag-tagged VHL alone, Flag-tagged VHL-nano/monobody fusion protein (see Figure 3 for a flow diagram describing the process).


      Figure 3. Schematic flow diagram illustrating the process by which target cell lines are infected with AdPROM and control retroviruses as outlined in Procedure C. Briefly, HEK-293 FT cells are sub-passaged and individually transfected with control or AdPROM constructs, along with the GAG/Pol and VSVG constructs required for retroviral production. Following incubation, retrovirus-containing medium from each cell dish is collected, filtered and applied to the target cells for 24 h. Infected target cells are selected in appropriate antibiotic-containing medium, and successful retrovirus integration is confirmed through Western blotting.

    3. Pipette 300 µl of serum-free Opti-MEM into two 1.5 ml Eppendorf tubes. To one of these tubes, add 6 µg of the cDNA of interest (e.g., Flag-VHL alone) in the pBABED puro vector, 3.2 µg of the pCMV-GAG/Pol, and 2.4 µg of the pCMV-VSVG. To the other tube, add 24 µl of 1 mg/ml PEI (diluted in 25 mM HEPES pH 7.5). A transfection control with pCMV5-GFP (10 µg) is also recommended.
    4. Allow the tubes to incubate for 5 min at room temperature (RT), before combining both tubes and incubating for a further 20 min at RT.
    5. Following the incubation period, add the resulting infection mix drop-wise on to the 10-cm dish of 70% confluent HEK-293 FT cells.
    6. Place cells in the 37 °C incubator and incubate for 16 h.
    7. Collect the medium from cells and dispose safely (by following appropriate disposal guidelines). Replace with fresh complete medium and incubate cells for 24 h.
    8. Collect the retrovirus-containing medium and filter through 0.45 µm sterile syringe filters into sterile tubes. Either store the filtered viral media at -80 °C, or add desired amount drop-wise onto the target cells (50-70% confluent at the time of infection) for infection. Typical dilutions range from 1:2 to 1:200 and need to be tested to achieve desired expression of AdPROM. Add polybrene (8 µg/ml, diluted in water) to aid infection. Dispose of the HEK-293 FT cells safely.
    9. Infect the target cells for 24 h.
    10. Remove the viral media, and wash the cells once in sterile PBS. Supply 10 ml of complete medium onto the target cells and allow them to recover for 24 h.
    11. Select the infected cells in appropriate antibiotic. For pBABED Puro vectors, puromycin (2 µg/ml) is applied to cells for 2 days (concentrations and length of antibiotic treatment sufficient to kill each target cell should be determined). As a control, include a dish of non-infected cells, to monitor the effectiveness of the antibiotic cytotoxicity.
    12. When control cells are all dead, collect the antibiotic-resistant cells and expand them for analysis by Western blotting.

  4. Testing the effectiveness of target protein degradation by constitutive expression AdPROM
    1. For lysis of AdPROM infected cells, wash cells twice in ice-cold PBS, and lyse cells in (200 µl-1 ml) ice-cold lysis buffer (see Recipes) using cell scrapers. Transfer extracts into 1.5 ml Eppendorf tubes and store on ice. Include non-infected wild type cells as a negative control.
    2. Following 10 min incubation on ice, clarify extracts by centrifugation (13,000 x g, 20 min, 4 °C).
    3. Collect the supernatant and perform a Bradford assay (Bradford, 1976) to quantify the protein concentration in each sample.
    4. Normalise the protein concentrations in each sample by diluting extracts in lysis buffer so that the protein concentration in each extract is the same.
    5. Add 5x SDS sample buffer (see Recipes) to each sample for a final concentration of 1x SDS sample buffer. Denature proteins by heating at 95 °C for 5 min. Samples can be stored at -20 °C for future use.
    6. Following a brief centrifugation (13,000 x g, 2 min, RT), extracts are ready to load onto SDS polyacrylamide gels.
    7. Resolve extracts (typically 20 µg protein per lane) by SDS-PAGE and transfer onto PVDF membranes. Include molecular weight markers on end lanes on both sides.
    8. For Western blotting, membranes are first blocked in 5% milk in TBS-T (1x) (see Recipes). Membranes are then incubated with primary antibodies diluted in either milk or BSA in TBS-T (1x) at 4 °C overnight, or for 1 h at RT (the dilution and milk vs. BSA need to be optimised for each antibody). To monitor target protein levels, use an antibody that recognises the POI, in addition to an antibody that recognises a house-keeping gene such as GAPDH or actin, for use as a loading control. Additionally, to confirm successful retrovirus integration, blot for the Flag-tag that was fused to the control and AdPROM retroviruses.
    9. Membranes are then washed in TBS-T, before incubation with HRP-conjugated secondary antibodies for 1 h at RT. Subsequently proteins of interest are detected by using ECL, and exposing the membranes to X-ray films for different exposure times.

  5. Adaptation of AdPROM for Tet-inducible POI degradation
    As described above (Procedure C-Procedure D) for constitutive POI degradation section, with the following changes:
    1. The pRetroX-Tet-On advanced system is a two-vector system, comprised of the pRetroX-Tet-On transactivator (G418 resistant) and the pRetroX-Tight gene of interest (puromycin resistant). These vectors are treated as per the pBABED puro vector system (6 µg added to the infection mix). One can either infect the target cells with both retroviruses at the same time and select for infection with both G418 and puromycin; or firstly infect target cells with the pRetroX-Tet-On transactivator, and select cells in G418 (200 µg/ml), and when non-infected cells are all dead, G418-resistant cells can be re-infected with the pRetroX-Tight AdPROM constructs and selected in puromycin (2 µg/ml).
    2. The viral dilutions for these retroviruses will need to be optimised. Ideally one wants to see no detectable AdPROM expression in the absence of Tet/doxycycline, but robust expression in the presence of Tet/doxycycline.
    3. For inducible AdPROM expression purposes, we recommend using 0.5-2 µg/ml doxycycline for the initial characterisation.
    4. Prior to cell lysis, expression of the AdPROM and control constructs will need to be performed, and ideally a Tet/doxycycline induction time course performed to monitor both AdPROM expression and target protein degradation.

  6. Degradation of proteins for which no nano/monobody is available
    In the event that no nano/monobody exists for your POI, we have shown that it is possible to first introduce an endogenous GFP knockin tag onto the gene encoding the POI using CRISPR/Cas9 gene editing technology, to enable targeting of the GFP-tagged target protein with high affinity anti-GFP nanobodies.
    Following successful GFP integration onto the target protein gene locus, the protocol for degradation of the GFP-tagged endogenous protein either constitutively or inducibly is the same as above (Procedure C-Procedure E).

Data analysis

  1. Have the retroviruses been integrated correctly? Blotting for the epitope-tagged VHL alone, nano/monobody alone, and the VHL-nano/monobody fusion protein will confirm successful AdPROM and control cDNA construct expression. Note that there will be a band shift corresponding to the molecular weight of the nano/monobody (around 100 amino acids) for the VHL-nano/monobody fusion protein relative to the VHL alone construct (see the original entries for representative Western blots highlighting the VHL mobility shift (Fulcher et al., 2016 and 2017)).
  2. Does AdPROM expression result in degradation of the POI? In uninfected cells and cells expressing VHL alone or the nano/monobody alone, there should be no degradation of the POI relative to the loading control. However, if target protein degradation is observed in cells expressing the VHL-nano/monobody polypeptide, with no detectable change in the levels of the loading control relative to the other samples, then the AdPROM system has been integrated successfully (see the original entries for representative Western blots highlighting the degradation of the POI with no concurrent proteolysis of the loading control protein (Fulcher et al., 2016 and 2017)).
  3. For the Tet-inducible system, if a time course experiment has been performed, blotting for VHL-nano/monobody polypeptide expression will determine the time for AdPROM expression. Blotting for the AdPROM-targeted POI will show the time taken for inducible proteolysis of the target protein. The degradation of the POI should succeed the AdPROM fusion protein expression.

Notes

  1. In some cases, expression of the nano/monobody alone retroviruses may inadvertently stabilise the target protein through binding to it and e.g., blocking ubiquitination sites present on the target protein.
  2. If, despite successful expression of the AdPROM system, no target protein degradation is observed, confirm nano/monobody:POI interaction through co-IP experiments. If the nano/monobody does not interact with the POI, or if the interaction is low-affinity, this could explain the lack of AdPROM-induced proteolysis of the target protein.
  3. For essential genes, constitutive AdPROM-mediated degradation of the target protein can still lead to lethality. In this instance, consider using the inducible AdPROM system to temporally control the proteolysis of the target protein.
  4. If the inducible AdPROM system is leaky, we recommend further dilution of the pRetroX-Tight gene of interest vector. Alternatively, replacing the FBS with Tet-free FBS may be helpful.

Recipes

  1. DNA loading buffer
    100 mg Orange G
    15 ml glycerol
    12 ml 0.5 M EDTA pH 7.5-8.0
    23 ml d/dH2O
  2. Lysis buffer
    50 mM Tris-HCl pH 7.4
    0.27 M sucrose
    120 mM NaCl
    1 mM EDTA pH 8.0
    1 mM EGTA pH 8.0
    1 mM sodium orthovanadate
    1 mM sodium glycerophosphate
    50 mM sodium fluoride
    10 mM sodium pyrophosphate
    1% Nonidet P-40
    0.1% β-mercaptoethanol (add fresh)
    Protease inhibitor cocktail tablet (add fresh)
    Diluted in d/dH2O
  3. Running buffer (10x)
    250 mM Tris
    1.9 M glycine
    1% (v/v) SDS
    Diluted in d/dH2O
  4. Transfer buffer (10x)
    250 mM Tris
    1.9 M glycine
    10% (v/v) final methanol
  5. Sample buffer (5x)
    312.5 mM Tris-HCl, pH 6.8
    50% (w/v) glycerol
    10% (w/v) SDS
    0.10% (w/v) bromophenol blue
    5% (w/v) β-mercaptoethanol
  6. TBS (10x)
    200 mM Tris
    1.5 M NaCl
    Diluted in d/dH2O
    pH to 7.6 with HCl
  7. TBS-T (1x)
    Dilute 10x TBS in d/dH2O
    Add 0.1% Tween-20
  8. TE buffer (10x)
    100 mM Tris-HCl pH 8.0
    10 mM EDTA pH 8.0
    Diluted in d/dH2O

Acknowledgments

We thank members of the Sapkota lab (P. Bozatzi, L. Hutchinson, C. Turnbull and S. Röth) as well as S. Nanda, I. Ganley, S. Virdee, A. Rojas-Fernandez and D. Alessi for helpful discussions, technical and experimental support during the development of the AdPROM system. We thank L. Fin, J. Stark and A. Muir for help with tissue culture. LJF is supported by the UK Medical Research Council PhD studentship. GPS is supported by the UK Medical Research Council (MC_UU_12016/3) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy, Dundee (GlaxoSmithKlien, Boehringer Ingelheim and Merck-Serono). We declare that we have no conflicts of interest or competing interests.

References

  1. Bosu, D. R. and Kipreos, E. T. (2008). Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div 3: 7.
  2. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54.
  3. Fulcher, L. J., Hutchinson, L. D., Macartney, T. J., Turnbull, C. and Sapkota, G. P. (2017). Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open Biol 7(5): 170066.
  4. Fulcher, L. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A. and Sapkota, G. P. (2016). An affinity-directed protein missile system for targeted proteolysis. Open Biol 6(10):160255.
  5. Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., Oeffinger, M., Nussenzweig, M. C., Fenyo, D., Chait, B. T. and Rout, M. P. (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 11(12): 1253-1260.
  6. Sambrook, J. and Russell, D. W. (2006). The Hanahan method for preparation and transformation of competent E. coli: High-efficiency Transformation. CSH Protoc 1;2006 (1)
  7. Schmidt, F. I., Lu, A., Chen, J. W., Ruan, J., Tang, C., Wu, H. and Ploegh, H. L. (2016). A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly. J Exp Med 213(5): 771-790.
  8. Sha, F., Gencer, E. B., Georgeon, S., Koide, A., Yasui, N., Koide, S. and Hantschel, O. (2013). Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains. Proc Natl Acad Sci U S A 110(37): 14924-14929.

简介

我们最近报道了一种针对内源性感兴趣蛋白(POI)的靶向蛋白水解的亲和指导PROtein导弹(AdPROM)系统(Fulcher等人,2016和2017)。 AdPROM由Von Hippel Lindau(VHL)蛋白组成,Cullin 2 E3连接酶底物受体(Bosu and Kipreos,2008),与识别细胞中靶蛋白的高亲和力多肽结合剂(如骆驼科纳米抗体)缀合。当在细胞中引入时,靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集,我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体(Sharm等人,2013; Fridy等人。,2014; Schmidt et al。,2016)。在此协议中,我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征,并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性,这些方法需要长时间的治疗并与脱靶效应相关联,而CRISPR / Cas9基因编辑并不总是可行的。
【背景】该协议使人们能够在哺乳动物细胞系中设计,构建和表达AdPROM VHL-nano /单体构建体以实现内源性POI的蛋白水解破坏。在原始条目中,我们通过使用识别绿色荧光蛋白(GFP)(Fridy等人,2014)或炎性蛋白质ASC(纳米抗体)的纳米抗体显示特定靶蛋白的几乎完全破坏Schmidt等人,2016)和两种不同的识别蛋白酪氨酸磷酸酶SHP2作为靶探针的单体(在许多人类癌症中)细胞系(Fulcher等人,2016和2017)。该方案提供了AdPROM构建体的产生,其在细胞中的表达以及监测目标蛋白质降解的细节,并且可以适用于任何纳米抗体和单体,用于POI的组成型和诱导型降解。该协议的重点不在于针对POI或CRISPR / Cas9基因组编辑(敲入POI上的GFP标签)的纳米/单体的产生,而是后面的步骤以利用AdPROM系统促进靶蛋白质的破坏。

关键字:AdPROM, 蛋白水解, VHL, Cullin2, 泛素化, 纳米抗体, 单抗体, CRISPR/Cas9

材料和试剂

  1. (Greiner Bio One International,目录号:771290; STARLAB INTERNATIONAL,目录号:S1111-1006; Greiner Bio One International,目录号:740295和Alpha Laboratories ,目录号分别为:LW1100)

  2. 15毫升猎鹰管(Greiner Bio One International,目录号:188271)

  3. 50毫升Falcon管(Greiner Bio One International,目录号:227261)
  4. 10-cm组织培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:172931)
  5. 微管(1.5毫升)(SARSTEDT,目录号:72.706.400)
  6. 0.45μm无菌注射器过滤器(Sartorius,目录号:16555-K)
  7. 0.22μm无菌注射器过滤器(Sartorius,目录号:16532-K)
  8. 用于组织培养的96孔板(Greiner Bio One International,目录号:655101)
  9. ImmobilonPPVDF膜(Merck,目录号:IPFL00005)
  10. 10毫升塑料注射器(BD,BD Biosciences,目录号:302188)
  11. 无菌一次性手术刀(斯旺莫顿,目录号:0503)
  12. X光胶片(柯尼卡美能达,APLUS)
  13. 用于逆转录病毒生产的HEK-293FT细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:R70007)
  14. 表达目标蛋白的感兴趣的细胞系
    注:我们在原始条目(Fulcher等,2016和2017)中使用了U2OS骨肉瘤,人胚胎肾HEK-293,腺癌A549和乳腺癌MDA-MB-231和MDA-MB-468细胞。
  15. 克隆级化学感受态DH5α细胞(使用Hanahan方法(Sambrook and Russell,2006)的修改版本内部制备)
  16. pBABED(Dundee修饰的pBABE载体)含有用于逆转录病毒生产的对照和AdPROM试剂的Puro FLAG载体
    注意:这些可以从MRCPPU获得 http:// mrcppureagents.dundee.ac.uk/reagent-catalogues (详情请参阅下面的AdPROM克隆程序)。
  17. pCMV-GAG / Pol(Cell Biolabs,目录号:RV-111)
  18. pCMV-VSVG(Cell Biolabs,目录号:RV-110)
  19. pRetroX-Tet-On用于Tet诱导性AdPROM表达的高级系统质粒(Takara Bio,Clontech,目录号:632104)
  20. 具有侧翼EcoRI / NotI位点的纳米/单体cDNA(在原始条目中使用的纳米/单体构建体的序列获自文献(Fulcher等, ,2016和2017))
  21. 限制性内切酶(FastDigest) - Bam HI,Eco RI,Dpn I和NotI(Thermo Fisher Scientific, Thermo Scientific TM,产品目录号分别为FD0054,FD0274,FD1703和FD0594)
  22. 超纯琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
  23. 测序寡核苷酸(0.025nM,Desalted)(Sigma-Aldrich)
  24. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  25. 快速DNA连接试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K1422)
  26. 氨苄青霉素(ForMedium,目录号:AMP25)
  27. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  28. PureLink TM HiPure质粒过滤器Maxiprep试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K210017)
  29. KOD热启动DNA聚合酶(Merck,目录号:71086-3)
  30. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  31. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  32. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,产品目录号:11960085)
  33. 胎牛血清(FBS)(Labtech,目录号:FCS-SA / 500)
  34. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030024)
  35. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  36. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985062)
  37. 细胞培养级胰蛋白酶(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  38. 聚乙烯亚胺(PEI)(Polysciences,目录号:24765)
  39. HEPES(Sigma-Aldrich,目录号:H4034)
  40. 聚凝胺(Sigma-Aldrich,目录号:107689)
  41. 嘌呤霉素(Sigma-Aldrich,目录号:P9620)
  42. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190169)
  43. 无脂奶粉(我们使用奇迹奶粉)
  44. 4-12%Bis / Tris梯度凝胶(Novex)
  45. 识别POI的一级抗体
    注:我们使用内部产生的抗体识别VPS34和FAM83G在第一个原始条目(Fulcher et al。,2016)。这两种抗体可根据要求提供,或从MRC-PPU试剂网站购买( http:// mrcppageagents .dundee.ac.uk /试剂目录)。对于第二个原始条目(Fulcher等人,2017),我们使用了抗SHP2(C端(Cell Signaling Technology,目录号:3397)和N端(Cell Signaling Technology,目录号:3752))和抗ASC(Martin Oeggerli,Adipogen,目录号:AL177)抗体。
  46. 识别GFP的一抗(ChromoTek的抗GFP抗体,目录号:3H9)和内部生成的抗GFP抗体,可从MRC-PPU试剂网站(






  47. 识别VHL的一抗(Cell Signaling Technology,目录号:68547)
  48. 识别管家基因的一抗(上样对照)(我们使用抗GAPDH(Cell Signaling Technology,目录号:2118))
  49. 牛血清白蛋白(BSA)粉末(Sigma-Aldrich,目录号:A7906)
  50. 增强化学发光(ECL)试剂(GE Healthcare,目录号:RPN2106)
  51. 二抗用于一抗鉴定
    注:我们使用抗羊IgG-HRP(Santa Cruz Biotechnology,目录号:sc-2770);和抗兔IgG,HRP-连接的(Cell Signaling Technology,目录号:7074)
  52. G418 /遗传霉素(Thermo Fisher Scientific,Gibco TM,目录号:10131035)
  53. 多西环素(盐酸盐)(Sigma-Aldrich,目录号:D3447)
  54. Orange G(Sigma-Aldrich,目录号:O3756)
  55. 甘油(VWR,目录号:24388.320)
  56. 乙二胺四乙酸(EDTA)(ForMedium,目录号:EDTA250)
  57. Tris(VWR,目录号:103157P)
  58. 蔗糖(VWR,目录号:27480.360)
  59. 氯化钠(NaCl)(VWR,目录号:27810.364)
  60. 乙二醇 - 双(β-氨基乙基醚)N,N,N',N' - 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  61. 原钒酸钠(Sigma-Aldrich,目录号:450243)
  62. β-甘油磷酸酯(Sigma-Aldrich,目录号:G9422)
  63. 氟化钠(Sigma-Aldrich,目录号:S7920)
  64. 焦磷酸钠(Sigma-Aldrich,目录号:P8010)
  65. Nonidet P-40替代品(Sigma-Aldrich,目录号:74385)
  66. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  67. 蛋白酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:11836170001)
  68. 甘氨酸(VWR,目录号:10119CU)
  69. 甲醇(VWR,目录号:20847.307)
  70. 十二烷基硫酸钠(SDS)(VWR,目录号:444464T)
  71. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  72. 用于pH调节的盐酸(HCl)(VWR,目录号:20252.335)
  73. 吐温20(Sigma-Aldrich,目录号:P1379)
  74. 明胶(来自猪皮)(Sigma-Aldrich,目录号:G2500)
  75. DNA加载缓冲液(见食谱)
  76. 裂解缓冲液(见食谱)
  77. 运行缓冲区(10倍)(见食谱)
  78. 转移缓冲区(10倍)(见食谱)
  79. 样品缓冲液(5x)(见食谱)
  80. TBS(10倍)(见食谱)
  81. TBS-T(1x)(见食谱)
  82. TE缓冲液(pH 8.0)(见食谱)

设备


  1. 水浴37°C和42°C
  2. 孵化器/摇床37°C(Infors)
  3. 台式离心机(4°C)
  4. 台式离心机(RT [室温])
  5. 500毫升锥形瓶
  6. 热循环仪(Thermo Fisher Scientific,Applied Biosystems,型号:ProFLEX PCR System)
  7. 涡(科学工业,模型:Vortex-Genie 2)
  8. 加湿培养箱细胞培养
  9. 用于组织培养的无菌罩,适用于2类工作
  10. 移液器(P2,P20,P200,P1000)
  11. 桌面热块
  12. 凝胶电泳仪(Peqlab)
  13. X光片开发商
  14. 高压灭菌器
  15. Nanodrop 1000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 1000)

程序

  1. AdPROM构建体在细胞中组成型表达的产生
    1. 插入物的制备
      1. 使用具有侧翼5'-EcoRI和3'-NotI位点的GeneArt(Life Technologies)合成具体的纳米/单抗结合结构域如下:GAATTCGCCATG-(纳米/单)-TGAGCGGCCGC。如果序列包含内部的Eco EcoRI或EcoI NotI位点,则引入沉默的变化以除去它们,使得侧翼位点是独特的,但是也可以根据细胞系被定位。
      2. 将冻干的GeneArt载体重新悬浮于50μld / dH 2 O(蒸馏/去离子H 2 O)中。
      3. 在37℃下将25μl重新悬浮的GeneArt载体与EcoRI和EcoI不同的I消化2h; (25μlDNA,10μlFastDigest绿色缓冲液,2μlEco RI,2μl非I,61μld / dH 2 O) 。
      4. 在1%琼脂糖凝胶(130V,35分钟)上运行完整消化(根据需要在相邻的孔之间分开)。两个乐队应该是可见的;较重的载体带应该在大约2.3-2.5Kb下运行,这取决于所提供的GeneArt主链,并且释放的插入带应该以合成的纳米/单体结合结构域的预期大小运行(参见图1,模拟DNA消化凝胶图像)。消化释放的cDNA插入物(小带)。对于DNA加载缓冲液(见食谱)。
      5. 使用QIAquick凝胶提取试剂盒根据制造商的说明书纯化消化的插入物,并在50μld / dH 2 O中洗脱。


        图1.模拟DNA琼脂糖凝胶突出显示在每个消化物中获得的预期产物。分子量(MW)以碱基对(bp)表示。 (步骤A1c)pMK-RQ AdPROM(GeneArt合成)×EcoRI RI +×NotI(上部条带=载体,2,286bp,下部条带=插入,307bp )。 2.(步骤A2b)pBABED P FLAG VHL 5×Gly不停止(DU54795)×EcoRI RI +不是I,5,830bp。 3.(步骤A2b)pBABED P FLAG(DU37983)×EcoRI RI + eI非5,176bp。 4.(步骤B2a)pRetroX-Tight Puro Eco EcoRI + NotI I,6,536bp。 5.(步骤B2i;步骤B3j)pRetroX-Tight Puro Bam HI + Not I,6,560bp。 6.(步骤B3c)VHL PCR xam Bam HI + em NotI,664bp。 7.(步骤B2e)纳米/单体PCR xml Bam HI +不是I,307bp。

    2. 载体的制备
      1. 消化接受载体:对于AdPROM构建体,切割pBABED Puro FLAG VHL(DU54795),并且对于纳米/单体只控制切割pBABED Puro FLAG(DU37983)。如果需要,还可以使用额外的VHL对照载体(DU54477)。所有组成型表达AdPROM试剂和载体可从MRC-PPU在线订购( http:// mrcppageagents。 dundee.ac.uk/reagent-catalogues )。
      2. 在37℃下消化每个接受载体的Eco EcoRI和EcoI Not I,持续2小时; (3-5μg载体,10μlFastDigest绿色缓冲液,2μlEco RI,2μl不是I,d / dH 2 O至100μl终体积)。 pBABED Puro FLAG VHL(DU54795)和pBABED Puro FLAG(DU37983)应分别产生5.9Kb和5.2Kb的单个片段(图1)。
      3. 在1%琼脂糖凝胶(130V,35分钟)上运行完整消化(根据需要在相邻的孔之间分开),切下剪切的载体条带(单个大条带)。
      4. 使用QIAquick凝胶提取试剂盒纯化消化的载体,并在50μld / dH 2 O中洗脱。
    3. 克隆,验证和Maxiprep
      1. 使用快速DNA连接试剂盒(5μl插入物,1μl载体,4μl5x快速DNA连接缓冲液,1μl连接酶,9μld / dH 2)将AdPROM插入物连接到空的和含VHL的接受载体 O)。
      2. 充分混合并在室温孵育5分钟。

      3. 加入5μl的连接反应到冰上的50μL感受态DH5α细胞,并离开2分钟。
      4. 将细胞移至42°C水浴45秒钟,然后置于冰上1分钟。
      5. 将转化管的全部内容物铺在含有100μg/ ml氨苄青霉素的LB(溶原培养基)琼脂平板上,并在37℃放置O / N(过夜)。
        注意:氨苄青霉素是一种缓效的抗生素,因此在接种前不需要回收步骤。
      6. 第二天早上,从平板挑取4-6个菌落,并分别加入15ml Falcons中的4ml LB(50μg/ ml氨苄青霉素)中。在37℃,220转/分钟的振荡培养箱中培养O / N。
      7. 生长16小时后,通过在4℃下以2,000×g离心10分钟来沉淀细胞。
      8. 根据制造商的说明,使用QIAprep Spin Miniprep试剂盒从每个沉淀中收获质粒DNA,并在50μld / dH 2 O中洗脱。
      9. 为每个设置一个小的20μL诊断消化,在37°C孵育1小时; (6μl质粒DNA,2μlFastDigest绿色缓冲液,0.1μlEco RI,0.1μl非I,11.8μld / dH 2 O )。
      10. 在1%琼脂糖凝胶上运行消化的样品,寻找AdPROM插入物的切除。两个乐队应该是可见的;较重的载体带应该在大约4.3Kb下运行,较小的释放插入带应该在合成的纳米/单体结合域的预期大小上运行。
      11. 使用引物pBABE F(5'-CCTCCTCTTCCTCCATCC)和pBABE R(5'-CCACACCTGGTTGCTGACTAATTGAG)的序列1或2个阳性。
      12. 用5ng来自确认克隆的质粒DNA转化50μlDH5α细胞,并在LB琼脂平板(100μg/ ml氨苄青霉素)上平板。
      13. 第二天早上挑一个菌落,在50ml Falcon管中接种10ml LB(50μg/ ml氨苄青霉素),并在37℃,220rpm下培养6-7小时。
      14. 将全部内容物加入含有150ml LB(50μg/ ml氨苄青霉素)的500ml锥形瓶中,并在37℃,220rpm下培养过夜。
      15. 使用PureLink TM HiPure Plasmid Filter Maxiprep Kit根据生产商的说明从培养物中收获质粒,并将纯化的质粒DNA洗脱在1ml TE缓冲液(pH 8.0)中。
      16. 使用NanoDrop仪器测量DNA浓度,为下游转染过程做准备;并通过限制性消化和DNA测序验证Maxi-prepped载体。

  2. 在细胞中产生用于四环素(Tet) - 诱导性表达的AdPROM构建体
    1. 纳米抗体的生成和引物设计
      1. 使用具有侧翼5'-EcoRI和3'-NotI位点的GeneArt(Life Technologies)合成特定的纳米/单体如下:GAATTCGCCATG-(纳米/单体)-TGAGCGGCCGC。如果序列包含内部的 Bam HI, RI或 Not I网站,则会引入无声更改以除去它们,
      2. pRetroX-Tight Puro目的载体的多克隆位点是有限的,因此有必要订购额外的引物来PCR扩增具有侧翼的Bam HI和Not I的AdPROM插入片段位点:gctaGGATCCGCCATG-(纳米/单体)-TGAGCGGCCGCgact(小写字母表示突出端以允许PCR产物的直接消化)。对于PCR引物推荐60℃的T m 如果低于那么相应地改变任何反应的退火温度。
      3. 设计寡核苷酸以扩增VHL(变体1, NM_000551.3 ) 3'-5xGLY接头,无终止密码子和侧翼5'-BamHI和3'-EcoRI限制性位点; gctaGGATCCGCC-(VHL)-GGTGGAGGCGGAGGTGAATTCgact(小写字母表示突出端以允许PCR产物的直接消化)。包含该克隆片段的保存载体(DU52906)可从MRC-PPU获得( http:// mrcppageagents .dundee.ac.uk / reagent-catalogs )。
    2. Tet诱导纳米/单体对照的产生
      1. 用BamHI和NotI消化载体pRetroX-Tight Puro以产生6.6Kb的单个片段(图1),如上所示纯化(步骤A2)并洗脱在50μld / dH 2 O中。
      2. 将冻干的GeneArt纳米/单体载体重悬于50μld / dH 2 O中。
      3. 使用KOD热启动DNA聚合酶(1μl模板[10ng /μlGeneArt cDNA],5μl10x PCR,用BamHI和NotI引物PCR扩增纳米/单体缓冲液,5μldNTP混合物[各2mM],3μl25mM MgSO 4,3μlDMSO,1.5μl各F&amp; R引物[10μM],1μlKOD聚合酶,29μld / DH <子> 2 O)。
      4. PCR:94℃2分钟,然后是94℃15秒,60℃30秒,和72℃25秒的30个循环,然后是72℃7分钟的单一延伸步骤。
      5. 在1%琼脂糖凝胶上用15μl上样缓冲液运行5μlPCR反应以确定成功。 PCR带应该匹配纳米/单体模板的大小(图1)。
      6. 使用QIAquick PCR纯化试剂盒根据制造商的说明清洁40μl确认的PCR反应(步骤B2d),但在85μld / dH 2 O中洗脱。
      7. 通过加入10μl10x FastDigest缓冲液,2μLBamHI,2μL不是I和1μLDpn I至10μL来切割整个清洁的PCR产物。来自步骤B2f的洗脱液。
        在37°C孵育至少2小时
      8. 重复PCR清除步骤,这次用50μld / dH 2 O洗脱以产生消化的AdPROM插入物。
      9. 除了在步骤A3i(图1)中使用 Bam HI和 Not <1>之外,在之前显示的克隆,验证和Maxiprep步骤之后,将AdPROM插入到pRetroX- ),在步骤A3k中用pBABE F(5'-CCTCCTCTTCCTCCATCC)和pRetroX R(5'-TGCTCCAGACTGCCTTGG)测序并在步骤A3n中接种250-400ml LB(50μg/ ml氨苄青霉素)。
    3. 产生Tet诱导的AdPROM载体
      1. 使用KOD热启动DNA聚合酶建立VHL PCR; (1μl模板[含VHL的载体,上面的部分A3]),5μl10x PCR缓冲液,5μldNTP混合物(各2mM),3μl25mM MgSO 4,3μlDMSO,1.5 μl每个F&amp; R引物(10μM),1μlKOD聚合酶,29μld / dH 2 O.
      2. PCR:94℃2分钟,然后94℃15秒,60℃30秒,72℃50秒30个循环,接着72℃7分钟的单个延伸步骤。
      3. 在1%琼脂糖凝胶上用15μl上样缓冲液运行5μlPCR反应以确定成功。 VHL和连接器的预期带宽应为0.7 Kb(图1)。
      4. 使用QIAGEN的QIAquick PCR纯化试剂盒根据生产商的说明清洁40μl确认的PCR反应(步骤B3b),但是在85μld / dH 2 O中洗脱。
      5. 切下整个清洁的PCR产物,加入10μL10x FastDigest缓冲液,2μL的乙烯HI,2μL的EcoRI RI和1μL的EM > Dpn I转移至来自步骤B3d的洗脱液。在37°C孵育至少2小时,然后重复PCR清除步骤,这次洗脱在50μLd / dH 2 O O产生消化的VHL插入。
      6. 在37℃下将25μl重新悬浮的GeneArt nano /单体载体与EcoRI和NotI在37℃消化2小时(25μlDNA,10μl10×FastDigest Green缓冲液,2μlEco RI,2μl非1μl,61μld / dH 2 O)。
      7. 在1%琼脂糖凝胶上运行完整的消化(根据需要在相邻的孔之间分开)并切除释放的cDNA插入物(小条带)。
      8. 使用QIAquick凝胶提取试剂盒进行纯化,并在50μld / dH 2 O中洗脱以产生AdPROM插入物。
      9. 通过使用快速DNA连接试剂盒将VHL和AdPROM插入片段同时插入到pRetroX-Tight Puro中,进行3向连接(参见图2) (2.5μlVHL插入片段(EcoBamHi / Eco EcoRI),2.5μlAdPROM插入片段(EcoRI / EcoRI / NotEmbol) I),1μlpRetroX-Tight Puro载体(BamHI / NotI),4μl5x快速DNA连接缓冲液,1μl连接酶,9μld / dH 2)。
      10. 按照先前显示的克隆,验证和Maxiprep步骤(A3部分)做一些小的修改;允许步骤A3b中的连接反应进行10分钟,在步骤A3c中向100μl细胞中加入10μl连接反应物,使用BamHi和不进行步骤E中的诊断消化物, I(图1),在步骤A3k中用pBABE F(5'-CCTCCTCTTCCTCCATCC)和pRetroX R(5'-TGCTCCAGACTGCCTTGG)进行测序,并在步骤A3m中接种250-400ml LB(50μg/ ml氨苄青霉素) >

        图2.示出了如步骤B3i中所述的典型的三路连接的示意图。该图显示了Tet诱导的pRetroX紧密质粒(参见程序E),然而,pBABED质粒的原理保持不变。

  3. 逆转录病毒生产和靶细胞感染的本构表达AdPROM系统
    1. 按照推荐的条件培养HEK-293FT细胞和所需的靶细胞系。对于HEK-293FT细胞,使细胞在补充有10%(v / v)FBS,2mM L-谷氨酰胺和1%(v / v)青霉素/链霉素(以下称为完全培养基)的DMEM中生长。细胞在5%CO 2下在潮湿的培养箱中保持在37℃。
    2. 在完全培养基中的每个10-cm培养皿中种植5×10 5个HEK-293FT细胞,并在37℃下在5%CO 2下潮湿的培养箱中培养过夜。第二天细胞应该〜70%融合。转染前,用9ml完全培养基更换培养基。一个约70%汇合的HEK-293FT细胞的10cm培养皿足以产生个体逆转录病毒。对于组成型表达AdPROM系统,我们推荐以下对照和AdPROM逆转录病毒:单独标记标签的纳米/单体,单独标记Flag的VHL,Flag-标记的VHL-纳米/单体融合蛋白(参见图3,描述过程的流程图)。


      图3.示意流程图,说明如程序C中所概述的,靶细胞系被AdPROM和对照逆转录病毒感染的过程。简言之,将HEK-293FT细胞亚传代并单独转染对照或AdPROM构建体,以及逆转录病毒生产所需的GAG / Pol和VSVG构建体。温育后,收集来自每个细胞培养皿的含有逆转录病毒的培养基,过滤并施用于靶细胞24小时。在含有适当抗生素的培养基中选择感染的靶细胞,通过蛋白质印迹证实成功的逆转录病毒整合。

    3. 移取300μl无血清Opti-MEM到两个1.5ml Eppendorf管中。向这些试管中的一个中加入6μg感兴趣的cDNA(例如,Flag-VHL单独)在pBABED puro载体中,3.2μg的pCMV-GAG / Pol和2.4μg的的pCMV-VSVG。向另一个管中加入24μl1mg / ml PEI(用25mM HEPES pH 7.5稀释)。
      pCMV5-GFP(10μg)的转染对照也被推荐
    4. 在室温(RT)下孵育5分钟,然后合并两个试管并在室温孵育20分钟。
    5. 在孵育期之后,将所得的感染混合物逐滴添加到70%融合的HEK-293FT细胞的10cm培养皿中。
    6. 将细胞放入37°C的培养箱中孵育16小时。
    7. 从细胞中收集培养基并安全处置(遵循适当的处置指南)。用新鲜的完全培养基代替,孵育细胞24小时。
    8. 收集含有逆转录病毒的培养基,并通过0.45μm无菌注射器过滤器过滤到无菌管中。将过滤的病毒培养基储存在-80°C,或将所需量滴加到靶细胞上(感染时50-70%融合)进行感染。典型的稀释度范围从1:2到1:200,需要进行测试以获得所需的AdPROM表达。加入聚凝胺(8μg/ ml,稀释于水中)以帮助感染。
      安全处置HEK-293 FT电池

    9. 感染靶细胞24小时
    10. 去除病毒的媒体,并在无菌PBS洗一次细胞。
      10毫升的完整培养基上的目标细胞,让他们恢复24小时。
    11. 选择合适的抗生素感染的细胞。对于pBABED Puro载体,将嘌呤霉素(2μg/ ml)应用于细胞2天(应确定足以杀死每个靶细胞的抗生素处理的浓度和长度)。作为对照,包括一盘未感染的细胞,以监测抗生素细胞毒性的有效性。
    12. 当对照细胞全部死亡时,收集抗生素抗性细胞并将其扩大用于Western印迹分析。

  4. 通过组成型表达AdPROM测试靶蛋白降解的有效性
    1. 为了裂解AdPROM感染的细胞,在冰冷的PBS中洗涤细胞两次,并使用细胞刮刀在(200μl-1ml)冰冷的裂解缓冲液(参见食谱)中裂解细胞。将提取物转移到1.5ml Eppendorf管中并储存在冰上。包括未感染的野生型细胞作为阴性对照。
    2. 在冰上孵育10分钟后,通过离心(13,000xg,20分钟,4℃)澄清提取物。
    3. 收集上清液并进行Bradford检测(Bradford,1976)以量化每个样品中的蛋白质浓度。
    4. 通过稀释裂解缓冲液中的提取物使每个样品中的蛋白质浓度标准化,使得每种提取物中的蛋白质浓度相同。
    5. 每个样品加入5x SDS样品缓冲液(见配方),最终浓度为1x SDS样品缓冲液。通过在95℃加热5分钟使蛋白质变性。样品可以储存在-20°C以备将来使用。
    6. 短暂离心(13,000×g,2分钟,RT)后,提取物准备上样到SDS聚丙烯酰胺凝胶上。
    7. 通过SDS-PAGE分离提取物(通常每道20μg蛋白质)并转移到PVDF膜上。
      包括分子量标记两端的车道
    8. 对于Western印迹,首先将膜在TBS-T(1x)中的5%牛奶中封闭(参见食谱)。然后将膜与在牛奶或BSA中在TBS-T(1x)中稀释的初级抗体在4℃温育过夜或在RT下1h(稀释和牛奶对BSA需要针对每种抗体进行优化)。为了监测目标蛋白质水平,除了识别管家基因(如GAPDH或肌动蛋白)的抗体外,还应使用识别POI的抗体作为上样对照。此外,为了确认成功的逆转录病毒整合,印迹与对照和AdPROM逆转录病毒融合的Flag标签。
    9. 然后在TBS-T中洗涤膜,然后在室温下与HRP缀合的第二抗体温育1小时。随后使用ECL检测感兴趣的蛋白质,并将膜暴露于X射线胶片以达到不同的暴露时间。

  5. AdPROM适应四环素诱导的POI降解
    如上所述(程序C-程序D)用于本构POI降解部分,具有以下变化:
    1. pRetroX-Tet-On高级系统是由pRetroX-Tet-On反式激活子(G418抗性)和pRetroX-Tight感兴趣基因(嘌呤霉素抗性)组成的双载体系统。这些载体按照pBABED puro载体系统处理(6μg添加到感染混合物中)。可以同时用两种逆转录病毒感染靶细胞,并选择G418和嘌呤霉素的感染;或首先用pRetroX-Tet-On反式激活物感染靶细胞,并选择G418(200μg/ ml)中的细胞,并且当未感染细胞全部死亡时,可用pRetroX-Tight重新感染G418抗性细胞AdPROM在嘌呤霉素(2μg/ ml)中构建和选择。
    2. 这些逆转录病毒的病毒稀释度需要进行优化。理想情况下,人们希望在不存在Tet /强力霉素的情况下看不到可检测的AdPROM表达,但是在Tet /强力霉素存在下的稳健表达。
    3. 为了诱导AdPROM表达的目的,我们推荐使用0.5-2μg/ ml多西环素进行初始表征。
    4. 在细胞裂解之前,需要进行AdPROM和对照构建体的表达,并且理想地进行Tet /强力霉素诱导时间进程以监测AdPROM表达和靶蛋白降解。

  6. 没有纳米/单体可用的蛋白质的降解
    如果您的POI没有纳米/单体存在,我们已经表明可以首先使用CRISPR / Cas9基因编辑技术将内源GFP敲入标签引入编码POI的基因,以使得能够靶向GFP-标签靶蛋白与高亲和力的抗GFP纳米抗体。
    在成功的GFP整合到目标蛋白质基因座上之后,组成性或诱导性降解GFP-标记的内源性蛋白质的方案与上述相同(程序C-程序E)。

数据分析

  1. 逆转录病毒是否被正确地整合?对单独的表位标记的VHL,单独的纳米/单体和VHL-纳米/单体融合蛋白的印迹将证实成功的AdPROM和对照cDNA构建体表达。注意,VHL-纳米单体融合蛋白相对于VHL单独构建体将具有对应于纳米/单体的分子量(约100个氨基酸)的条带漂移(参见代表性Western印迹的原始条目,突出显示VHL流动性转变(Fulcher et。,2016和2017))。
  2. AdPROM表达是否导致POI降解?在单独表达VHL或单独表达纳米/单体的未感染细胞和细胞中,相对于加载对照,POI不应该降解。然而,如果在表达VHL-nano /单体多肽的细胞中观察到靶蛋白质降解,相对于其他样品,加载对照水平没有可检测的变化,则AdPROM系统已成功整合(参见原始条目代表性的蛋白质印迹强调了POI的降解,而没有加载控制蛋白的同时蛋白水解(Fulcher等人,2016和2017))。
  3. 对于Tet诱导系统,如果已经进行了时间过程实验,VHL-纳米/单体多肽表达的印迹将决定AdPROM表达的时间。针对AdPROM靶向的POI的印迹将显示靶蛋白可诱导蛋白水解所花费的时间。
    POI的降解应该能够成功实现AdPROM融合蛋白的表达

笔记

  1. 在一些情况下,单独的纳米/单抗逆转录病毒的表达可能无意中通过与目标蛋白结合而稳定目标蛋白,例如阻断存在于目标蛋白上的遍在蛋白化位点。
  2. 如果尽管AdPROM系统成功表达,但没有观察到目标蛋白降解,通过共IP实验确认纳米/单体:POI相互作用。如果纳米/单体不与POI相互作用,或者如果相互作用是低亲和性的,这可以解释缺少AdPROM诱导的靶蛋白的蛋白水解。
  3. 对于必需基因,组成型AdPROM介导的靶蛋白降解仍然可能导致致命性。在这种情况下,考虑使用可诱导的AdPROM系统来暂时控制靶蛋白的蛋白水解。
  4. 如果可诱导的AdPROM系统泄漏,我们建议进一步稀释pRetroX-Tight目的载体。或者,用无Tet的FBS取代FBS可能有帮助。

食谱

  1. DNA加载缓冲液
    100毫克橙G
    15毫升甘油
    12ml 0.5M EDTA pH 7.5-8.0
    23ml d / dH 2 O 0
  2. 裂解缓冲液
    50 mM Tris-HCl pH 7.4
    0.27 M蔗糖
    120 mM NaCl
    1 mM EDTA pH 8.0
    1 mM EGTA pH 8.0
    1 mM原钒酸钠
    1 mM甘油磷酸钠
    50 mM氟化钠
    10 mM焦磷酸钠
    1%Nonidet P-40
    0.1%β-巯基乙醇(添加新鲜)
    蛋白酶抑制剂鸡尾酒片(添加新鲜)
    在d / dH 2 O中稀释
  3. 运行缓冲区(10x)
    250 mM Tris
    1.9 M甘氨酸
    1%(v / v)SDS
    在d / dH 2 O中稀释
  4. 传输缓冲区(10x)
    250 mM Tris
    1.9 M甘氨酸
    10%(v / v)最终甲醇
  5. 样本缓冲区(5x)
    312.5mM Tris-HCl,pH6.8
    50%(w / v)甘油
    10%(w / v)SDS
    0.10%(w / v)溴酚蓝
    5%(w / v)β-巯基乙醇
  6. TBS(10x)
    200 mM Tris
    1.5 M NaCl
    在d / dH 2 O中稀释 用HCl将pH值降至7.6
  7. TBS-T(1x)
    在d / dH 2 O 0中稀释10倍TBS 添加0.1%Tween-20
  8. TE缓冲液(10x)
    100 mM Tris-HCl pH 8.0
    10 mM EDTA pH 8.0
    在d / dH 2 O中稀释

致谢

我们感谢Sapkota实验室的成员(P. Bozatzi,L. Hutchinson,C. Turnbull和S.Röth)以及S. Nanda,I. Ganley,S. Virdee,A. Rojas-Fernandez和D. Alessi讨论,在AdPROM系统开发过程中的技术和实验支持。我们感谢L. Fin,J. Stark和A. Muir对组织培养的帮助。 LJF得到英国医学研究理事会博士学位的支持。 GPS由英国医学研究委员会(MC_UU_12016 / 3)和支持信号转导治疗部门的制药公司Dundee(GlaxoSmithKlien,Boehringer Ingelheim和Merck-Serono)提供支持。我们宣布我们没有利益冲突或利益冲突。

参考

  1. Bosu,D.R。和Kipreos,E.T。(2008)。 滞蛋白-RING泛素连接酶:全球监管和激活周期 细胞股利 3:7.
  2. Bradford,M.M。(1976)。 利用蛋白质 - 染料结合原理定量微克蛋白质量的快速而灵敏的方法。 Anal Biochem 72:248-54。
  3. Fulcher,L.J.,Hutchinson,L.D.,Macartney,T.J.,Turnbull,C。和Sapkota,G.P.(2017)。 通过亲和导向蛋白质导弹系统瞄准内源蛋白降解 打开生物学7(5)。
  4. Fulcher,L.J.,Macartney,T.,Bozatzi,P.,Hornberger,A.,Rojas-Fernandez,A。和Sapkota,G.P。(2016)。 针对蛋白水解的亲和定向蛋白导弹系统 Open Biol < / em> 6(10)。
  5. Fridy,PC,李,Y.,基冈,S.,汤普森,MK,诺德尔曼,一,赛德,JF,Oeffinger,M.,Nussenzweig,MC,Fenyo,D.,蔡特,BT和溃败,MP(2014 )。 快速制作多功能纳米抗体曲目的强大管道 Nat Methods < / em> 11(12):1253-1260。
  6. Sambrook,J。和Russell,D.W。(2006)。 汉汗方法的准备和改造主管。大肠杆菌:高效转化。 CSH Protoc 1; 2006(1)
  7. Schmidt,F. I.,Lu,A.,Chen,J. W.,Ruan,J.,Tang,C.,Wu,H.和Ploegh,H.L。(2016)。 识别该适配器ASC单结构域抗体片段定义了在炎性组件ASC结构域的作用。Exp J Exp Med 213(5):771-790。
  8. Sha,F.,Gencer,E. B.,Georgeon,S.,Koide,A.,Yasui,N.,Koide,S.and Hantschel,O。(2013)。使用高度特异性monobody抑制剂到SHP2 SH2结构域的BCR-ABL信令网络的解剖。(Proc Natl Acad Sci USA)110(37):14924-14929。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Macartney, T. J., Sapkota, G. P. and Fulcher, L. J. (2017). An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins. Bio-protocol 7(22): e2614. DOI: 10.21769/BioProtoc.2614.
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