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Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
mbSUS酵母双杂系统检测蛋白质的相互作用   

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Abstract

The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K+ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)

Keywords: mbSUS(mbSUS), Yeast(酵母), Mating(交配), Split-ubiquitin(分裂泛素), Protein-protein interaction(蛋白质 - 蛋白质相互作用), Gateway(Gateway)

Background

Figure 1 is an overview of the mbSUS assay. The ubiquitin moiety is split into two halves, and the N-terminal half is mutated (NubG) to avoid reassembly. The C-terminal half of the ubiquitin moiety (Cub) is linked with the transcription reporter complex PLV (Protein A-LexA-VP16). The fusion of two proteins (X and Y) to NubG and CubPLV, respectively, yields a system for protein-protein interaction analysis. After transformation, the yeast strain THY.AP5 contains the NubG-X fusion protein, while yeast strain THY.AP4 contains the Y-CubPLV fusion protein. After mating, in the diploid yeast, if proteins X and Y interact with each other, a functional ubiquitin will be reassembled which leads to cleavage of the PLV. The released transcription protein complex PLV switches on reporter genes (ADE2, HIS3) and allows yeast growth on selective media.


Figure 1. The split-ubiquitin system. A. The Ubiquitin is split into two halves, the N-terminal wild type half (NubWt; NubI) and the C-terminal half (Cub). Reassembly of NubWt and Cub leads to the release of the transcription reporter complex Protein A-LexA-VP16 (PLV) by ubiquitin-specific proteases (USPs). B. Mutating the isoleucine at position 13 of the N-terminal half to glycine yields the NubG protein, which inhibits the spontaneous reassembly of ubiquitin. In diploid yeast, NubG-X and Y-CubPLV fusion proteins are produced. If X and Y do not interact, no functional ubiquitin is formed, and the yeast cannot grow on selective media as shown in a small image on top-right. C. If X interacts with Y, then NubG and Cub can reassemble as a functional ubiquitin, which leads to the release of PLV. The PLV activates the reporter genes (ADE2, HIS3) synthesising ADE and HIS, which allows yeast growth on the selective media without these chemicals as shown in a small image on top-right.

Materials and Reagents

  1. Sterile toothpick
  2. Round bottom polystyrene tubes, 14 ml (purchased as sterile) (Corning, catalog number: 352051 )
  3. Blue screw cap tubes, 50 ml (purchased as sterile; Cellstar)
  4. Screw cap tubes, 2 ml (purchased as sterile; SARSTEDT)
  5. PCR tubes, 0.2 ml, flat cap (Sterilized by autoclaving) (STARLAB INTERNATIONAL, catalog number: I1402-8100 )
  6. Nitrocellulose transfer membranes, BioTraceTM NT (Pall, catalog number: 66485 )
  7. 1.5 ml screw cap tube
  8. Minisart® syringe filter, non-pyrogenic, 0.2 µm (Sartorius)
  9. Syringe filter w/0.45 µm polyethersulfone membrane (VWR, catalog number: 28145-503 )
  10. Blotting paper (VWR, Blotting pad 707)
  11. Petri dishes, 55 mm (purchased as sterile) (Greiner Bio One International, catalog number: 628102 )
  12. Square Petri dishes, 120 x 120 mm (purchased as sterile) (Greiner Bio One International, catalog number: 688102 )
  13. Filter tips 10, 200, 1,000 μl (Biosphere® Plus, for micropipettes, SARSTEDT)
  14. Saccharomyces cerevisiae yeast strains used in the mbSUS assay (Table 1)

    Table 1. Yeast strains used for mbSUS assay
    Name
    Genotype
    Function
    Reference
    THY.AP4
    MATa;
    ade2-, his3-, leu2-, trp1-,
    ura3
    -;
    lexA::ADE2,
    lexA::HIS3, lexA::lacZ

    Bait: carrying the Y-
    CubPLV in vector
    pMetYC-Dest
    (Obrdlik et al., 2004)
    THY.AP5
    MATα;
    URA3;
    ade2-, his3-, leu2-, trp1- 
    Prey: carrying the
    NubG-X in vector
    pNX35-Dest or NubWt
    (Obrdlik et al., 2004)

  15. Destination vectors used in the mbSUS assay (Table 2)

    Table 2. The destination vectors used for mbSUS assay
    Vector name
    Promotor
    Gateway site
    Function
    Reference
    pMetYC-Dest
    met25
    attR1, attR2
    Fusion protein with C-terminal
    CubPLV as bait; synthesis of
    LEU in yeast
    (Grefen et al., 2009)
    pNX35-Dest
    ADH1
    attR1, attR2
    Fusion protein with N-terminal
    NubG as prey; synthesis of
    TRP in yeast 
    (Grefen and Blatt, 2012)

  16. Donor vector: pDONR207 vector (Thermo Fisher Scientific) or other donor vectors contain attP1, attP2 Gateway cassette
  17. Gateway® clonase enzyme:
    1. BP Clonase®II Enzyme Mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11789020 )
    2. LR Clonase®II Enzyme Mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11791020 )
  18. Protein ladder (Pageruler Plus Prestained Protein Ladder, Thermo Fisher Scientific)
  19. Western blot signal detection kit (SuperSignal West Dura Chemiluminescent Substrate) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 37071 )
  20. Antibody solutions
    1. Primary: anti-HA (1:20,000, Anti-HA high-affinity Rat monoclonal antibody) (Roche Diagnostics, catalog number: 11867423001 ) or anti-VP16 (1:20,000, Anti-VP16 tag antibody in Rabbit) (Abcam, catalog number: ab4808 )
    2. Secondary: anti-rabbit HRP (1:20,000, goat anti-rabbit IgG-HRP) (Thermo Fisher Scientific, InvitrogenTM, catalog number: G-21234 ) or anti-rat HRP (1:20,000, Rabbit anti-Rat IgG H&L [HRP]) (Abcam, catalog number: ab6734 )
  21. Methionine
  22. Glycerol (Fisher Scientific, catalog number: 10795711 )
  23. Peptone (FormediumTM, catalog number: PEP02 )
  24. Glucose (Fisher Scientific, catalog number: 10141520 )
  25. Yeast extract (FormediumTM, catalog number: YEA02 )
  26. Oxoid agar (Agar No.1) (Oxoid, catalog number: LP0011 )
  27. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
  28. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L4158 )
  29. De-ionized water
  30. Acetic acid (Sigma-Aldrich, catalog number: 1.00063 )
  31. Salmon sperm DNA (ssDNA) (Sigma-Aldrich, catalog number: 31149 )
  32. Polyethylene Glycol 3350 (Spectrum Chemical Manufacturing, catalog number: Po125-500gm )
  33. YNB without ammonium sulphate, without potassium (MP Biomedicals, catalog number: 114029622 )
  34. Potassium dihydrogen orthophosphate (Fisher Scientific, catalog number: 10783611 )
  35. CSM-ADE-HIS-LEU-MET-TRP-URA (powder) (MP Biomedicals, catalog number: 114560222 )
  36. Agar
  37. Adenine sulphate (Sigma-Aldrich, catalog number: A3159 )
  38. Uracil (Sigma-Aldrich, catalog number: U1128 )
  39. L-leucine (Sigma-Aldrich, catalog number: L8912 )
  40. L-tryptophane (Sigma-Aldrich, catalog number: T4196 )
  41. L-histidine (Sigma-Aldrich, catalog number: H3911 )
  42. L-methionine (Sigma-Aldrich, catalog number: M5308 )
  43. Sodium dodecyl sulfate (SDS) (VWR, catalog number: 442444H )
  44. Urea (Sigma-Aldrich, catalog number: U5378 )
  45. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43817 or 43815 )
    Note: The product “ 43817 ” has been discontinued.
  46. Acrylamide (Sigma-Aldrich, catalog number: A8887 )
  47. Bromophenol blue
  48. Ammonium persulfate (Fisher Scientific, catalog number: 10020020 )
  49. TEMED (Sigma-Aldrich, catalog number: T9281 )
  50. Ammonium sulphate (VWR, catalog number: 21333.296 )
  51. Tris (Fisher Scientific, catalog number: BP152-1 )
  52. Glycine (Fisher Scientific, catalog number: 10070150 )
  53. Methanol (Sigma-Aldrich, catalog number: 34860 )
  54. Ponceau S (Sigma-Aldrich, catalog number: P3504 )
  55. Sodium chloride (VWR, catalog number: 27810.295 )
  56. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  57. Tween® 20 (Sigma-Aldrich, catalog number: P1379 )
  58. Milk powder (Marvel, Iceland, UK)
  59. YPD media (see Recipes)
  60. LiAc solution (see Recipes)
  61. ssDNA solution (see Recipes)
  62. PEG solution (see Recipes)
  63. CSM-minimal media (see Recipes)
  64. Reagents for auxotrophy selection (see Recipes)
  65. SCM-auxotrophy selection media (see Recipes)
  66. ‘Lyse & Load’ buffer (see Recipes)
  67. 10% SDS-PAGE separation gel (see Recipes)
  68. 5% SDS-PAGE stacking gel (see Recipes)
  69. Bjerrums buffer (see Recipes)
  70. Ponceau S solution (see Recipes)
  71. TBS solution and TBS-Tween solution (see Recipes)
  72. Running buffer (see Recipes)
  73. Blocking solution (see Recipes) 

Equipment

  1. Incubator shaker for yeast growth in liquid culture (Sartorius, model: BS1 )
  2. Glass flask, 500 ml (sterilized by autoclaving) (Corning, PYREX®, catalog number: 4980-500 )
  3. High speed refrigerated microcentrifuge (Eppendorf, model: 5417 R )
  4. Refrigerated centrifuge for large volume liquid samples (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM RT )
  5. PCR machine (VWR, PEQLAB, model: peqSTAR 96 Universal Gradient )
  6. Pipette  
  7. Incubator for yeast growth on solid media plates (Gallenkamp, model: Ipr150 )
  8. Ultrasonic disintegrator (MSE, model: Soniprep 150 MSS150.CX , 9.5 mm probe)
  9. Biophotometer (Eppendorf, model: Biophotometer Plus 6132 )
  10. SDS-PAGE gel electrophoresis and blotting boxes (Bio-Rad Laboratories, models: Power PAC 1000 and Mini-PROTEAN Tetra Cell )
  11. Shaker for Western blot (Stuart, model: Stuart STR9 Gyro rocker )
  12. Semi-dry blotting system (VWR, PEQLAB, model: PerfectBlueTM Semi-Dry Blotter )
  13. Western blot imaging platform (Vilber, model: Fusion spectra )
  14. Vortex (Scientific Industries, model: Vortex-Genie 2 )
  15. Imaging system
  16. Autoclave

Procedure

  1. Molecular biology
    Gateway® Technology (Thermo Fisher Scientific, Germany) is used to create plasmids for the yeast mbSUS assay following the manufacturer’s instructions. The unique attB1 and attB2 sites (B1 5’-3’: ggggacaagtttgtacaaaaaagcaggct; B2 5’-3’: ggggaccactttgtacaagaaagctgggt) are included in the forward and reverse primers as overhanging sequences, respectively. To fuse the PCR product in the proper reading frame with an N-terminal tag, the forward primer must include two additional nucleotides. Depending on the destination vector, a stop codon is included in the reverse primer or provided on the vector itself.
    Subsequent PCR products are inserted by BP reactions into the pDONR207 vector to yield Entry clones that are verified by DNA sequencing. Gateway Destination clones, pNX35-Dest for prey (Grefen and Blatt, 2012) and pMetYC-Dest for bait (Grefen et al., 2009), are generated by using LR Clonase® II in LR reactions according to the manufacturer’s instructions.

  2. Yeast transformation
    The yeast transformation is performed under sterile condition. Prepare fresh competent yeast for each transformation.
    1. Pick THY.AP4 and AP5 yeast from long-term stock by a sterile toothpick or pick one colony of yeast from a stock plate (see Note 7) and inoculate 5 ml YPD in 14 ml sterile round bottom tubes and incubate in a shaker overnight at 200 rpm, 28 °C.
    2. Inoculate 45 ml of YPD with the 5 ml of overnight culture in autoclaved 500 ml flasks and incubate with shaking at 200 rpm, 28 °C for 3-5 h until OD600 0.6-0.8.
    3. Harvest cells by centrifugation (10 min at 2,000 x g, 4 °C) in 50 ml blue screw cap tubes and discard the supernatant.
    4. Wash cells with 20 ml of sterile water, centrifuge again, and discard the supernatant.
    5. Resuspend the cells with 1.8 ml of 0.1 M LiAc and transfer to a 2 ml Eppendorf tube, spin down (30 sec at 2,000 x g, 4 °C), and discard the supernatant.
    6. Add an appropriate amount of 0.1 M LiAc (multiply the number of transformations by 20 µl) and incubate at room temperature for 30 min.
    7. Meanwhile, prepare sterilized PCR tubes with 10 µl of ssDNA and 5 µl of plasmid DNA (Destination clones, > 100 ng/μl) for each transformation.
    8. Prepare a master mix: mix 70 µl of PEG solution, 10 µl of 1 M LiAc, and 20 µl of competent yeast (from step B6) for each transformation.
    9. Mix the master mix with the DNA mixture from step B7.
    10. In a PCR machine, incubate at 30 °C for 30 min, then gently mix the mixture by pipette and heat shock cells at 43 °C for 30 min.
    11. Spin down the yeast at 2,000 x g for 1 min, and discard the supernatant by pipetting it carefully off the pelleted cells.
    12. Wash the cells with 100 µl of sterile water, spin down, and discard the supernatant.
    13. Resuspend cells in 50 µl of sterile water and plate on the appropriate selective media.
      1. THY.AP4 carrying bait vector plates on CSM-LM solid medium.
      2. THY.AP5 carrying prey vector plates on CSM-TUM solid medium.
    14. Incubate for 48-72 h at 28 °C. At least 100 colonies are expected in each plate.

  3. mbSUS assay
    The mbSUS assay is performed under sterile conditions.
    1. Pick 5-15 colonies of transformed THY.AP5 yeast carrying the prey construct and inoculate in 3 ml CSM-TUM media. Grow overnight in the incubator shaker at 200 rpm, 28 °C.
    2. Pick 5-15 colonies of transformed THY.AP4 yeast carrying the bait construct and inoculate in 3 ml CSM-LM media. Grow overnight in the incubator shaker at 200 rpm, 28 °C.
    3. Reserve 750 µl from each bait and prey yeast culture to store as a stock culture (see Note for yeast stock preparation).
    4. Harvest yeast cells by centrifugation at 2,000 x g for 1 min at room temperature and resuspend the pellets in YPD media (for each mating 20 µl of YPD media should be calculated).
    5. Mix 20 µl of each yeast (carrying bait) and yeast (carrying prey) in sterilized PCR tube and immediately drop 4 µl of each mated yeast onto YPD plate. Incubate overnight at 28 °C.
    6. Transfer the mated yeast from the YPD plate to a CSM-LTUM plate with a sterile toothpick (Figure 2A). Grow overnight at 28 °C.


      Figure 2. Example of a mbSUS assay. A. Yeast is transferred from the YPD plate to a CSM-LTUM plate with a sterile toothpick. B. Yeast is dropped on the CSM-LTUMAH plate with black spots on paper. C. Diploid yeast expressing KAT1-CubPLV as bait with NubG-X fusions of VAMP721, VAMP723, and the controls (negative: NubG; positive: NubWt) as prey were spotted onto different media as indicated. CSM-LTUM was used to verify the presence of both bait and prey vectors. The addition of different concentration of methionine (Met) to CSM-LTUMAH was used to verify interaction when K+ channel-CubPLV expression was suppressed. Diploid yeast was dropped at 1.0 and 0.1 OD600 in each case. Incubation time was 24 h for CSM-LTUM plate and 72 h for CSM-LTUMAH plates. Western blot analysis of the diploid yeast used in mbSUS was carried out with commercial HA antibody for the VAMP fusions and VP16 antibody for the K+ channel fusions. Ponceau S stains were used as blotting/loading control (right).

    7. Pick the mated yeast colony of each sample from the CSM-LTUM plate and inoculate in 3 ml liquid CSM-LTUM media, grow overnight in the incubator shaker at 200 rpm, 28 °C.
    8. Inoculate 1 ml of the mated cells into 3 ml of fresh CSM-LTUM media and grow at 200 rpm, 28 °C for 4-6 h for later Western blot analysis.
    9. Determine OD600 of yeast cells from step C7.
    10. Harvest 100 µl of remaining cells from step C7 by centrifugation at 2,000 x g, 1 min at room temperature and dilute them to OD600 1.0 and 0.1 respectively in sterile water.
    11. Apply 4 µl drops of each dilution on CSM-LTUM and CSM-LTUMAH plates with different methionine concentrations (0.5, 5, 50, and 500 µM; Figure 2B), grow them at 28 °C. Check for growth after 1 day and 3 days growth on CSM-LTUM plate and CSM-LTUMAH plate respectively. Figure 2C shows an example of a mbSUS assay which indicates an interaction between VAMP721 and KAT1 K+ channel, and no interaction between VAMP723 and KAT1 (Zhang et al., 2015). Diploid yeast containing NubWt as prey serves as the positive control and yeast containing NubG as prey is the negative control. In a good mbSUS assay, the sample of negative control should show weak or no yeast growth on CSM-LTUMAH plates, while the yeast grows well in the positive control sample (Figure 2C).

  4. Yeast protein extraction for western blot analysis
    1. Determine the OD600 of the yeast culture.
    2. Harvest 2 ml of the culture and resuspend in ‘Lyse & Load’ buffer at a final OD600 of 100 for each sample. The volume of ‘Lyse & Load’ buffer used is calculated like this: e.g., the 1:10 diluted culture measurement yields an OD600 of 0.5, which is equivalent to an OD600 of 5 for 1 ml of culture. Harvest 2 ml of the culture yields OD600 of 10. Then resuspend them in 0.1 ml of ‘Lyse & Load’ buffer can get a final OD600 of 100 in the sample.
    3. Lyse the yeast with an ultrasonic disintegrator (amplitude setting 10 for 20 sec, keep samples on ice).
    4. Incubate for 10 min at 65 °C.
    5. Vortex for 10 sec, spin down the lysate at full speed for 30 sec and transfer the supernatant to a fresh tube. Then sample can be used for Western blot analysis.

  5. Western blot analysis
    1. Load protein ladder and 10-15 μl of each sample on an SDS-PAGE gel.
    2. Run gel with appropriate conditions and transfer proteins to a nitrocellulose membrane (e.g., by semidry transfer).
    3. Block the membrane for 60 min in the blocking buffer at room temperature.
    4. Transfer the membrane to the primary antibody solution; incubate for at least 3 h at room temperature or overnight at 4 °C.
    5. Wash 3 times with 1x TBS-Tween, 10 min each wash.
    6. Transfer the membrane into the secondary antibody solution; incubate for at least 3 h at room temperature or overnight at 4 °C.
    7. Wash 3 times with 1x TBS-Tween, 10 min each wash.
    8. Incubate the membrane with the chemiluminescent substrate for 5 min in darkness.
    9. Develop the membrane and take images with an imaging system.
    10. Wash membrane several times with distilled water to remove the chemiluminescent substrate.
    11. Incubate the membrane in the Ponceau S solution for 5 min.
    12. Wash the membrane several times with distilled water until the red bands are clearly visible.
    13. Scan Ponceau S image as blotting/loading control.

Notes

  1. When using proteins fusions, the C-terminus of the native protein with the Y-CubPLV fusion should be located in the cytosol and the NubG-X fusion of the prey should also be available in the cytosol for effective interactions to take place. Reassembly of Cub and Nub then leads to cleavage of the PLV moiety and allows this transcription factor to move into the nucleus.
  2. Macromolecules smaller than 40 kDa can passively diffuse through the nuclear pore, and some larger particles are also able to pass through the large diameter of the pore (Marfori et al., 2011). So the bait protein should normally be anchored to a membrane. Soluble bait fusions could otherwise move into the nuclear and activate the reporter genes independent of any interaction and PLV cleavage. Preys fusions need not be membrane anchored.
  3. Expression of fusion proteins in the bait vector pMetYC-Dest is under control of the met25 promoter which suppresses expression in the presence of methionine in the culture media (Grefen et al., 2009). However, our experience of mbSUS assay shows that the yeast growth on media without external methionine is slower than the yeast on media adding 5 μM methionine. To overcome this problem, it is recommended to add 0.5 μM methionine to the media. Higher concentrations, up to 1 mM Met, are used to test for specificity in interaction by reducing the bait expression.
  4. Before yeast dropping, it is recommended to allow media plates to dry. Allow the surface of the plate to dry for 30 min under sterile conditions. The dry surface of the solid media in plate helps to keep the yeast-spot round.
  5. When dropping yeast, it is recommended to print a dropping scheme with black spots on paper and stick it under the plate, as shown in Figure 2B left.
  6. For Western blot analysis, we recommend that the yeast is not cultured overnight. Add 3 ml fresh media to a 1 ml yeast culture from the previous overnight growth and allow the culture to grow for a further 4-6 h before harvesting. This approach avoids the possibility of senescent yeast in the culture which may introduce degraded protein and lead to poor Western blot results.
  7. To prepare yeast stock for long-term storage, pipette 750 µl of fresh overnight yeast culture (from step C1 or C2) and add 250 µl of sterile 60% glycerol to a 1.5 ml screw cap tube, tightly close the cap and mix before freezing at -80 °C. It is recommended to refresh glycerol stocks every 6 months and avoid repeated freeze-thaw cycles of the glycerol stock. To recover yeast cells from a glycerol stock, pick frozen yeast cells using a sterile toothpick, drop and spread onto the appropriate selective media solid plate, then incubate at 28 °C for 24-48 h. Yeast colonies on selective media are ready to use for mbSUS assay, and the yeast colonies can be stored at 4 °C for up to 1 month. To prepare a yeast stock for short-term storage, pipette 5-10 µl of fresh overnight yeast culture (from step C1 or C2) or pick frozen yeast cells from glycerol stock; and drop cells on appropriate selective solid media. Culture yeast for 24-48 h at 28 °C, then store at 4 °C for up to1 month.

Recipes

  1. YPD media
    2% peptone
    2% glucose
    1% yeast extract
    2% agar
    Adjust pH to 6.0 with 1 N KOH
    Sterilize by autoclaving and store at 4 °C
  2. LiAc solution (1 M or 0.1 M)
    Dissolve lithium acetate in de-ionized water. Adjust the pH to 7.5 with acetic acid, sterilize by filtration (Minisart® Syringe Filter) and store at room temperature
  3. ssDNA solution
    Dissolve 10 mg/ml ssDNA in de-ionized water, sterilize by filtration (Minisart® Syringe Filter) and boil for 5 min following cooling on ice before use. Store at -20 °C
  4. PEG solution
    Dissolve PEG 3350 in de-ionized water to a final concentration of 50% (w/v), sterilize by filtration (0.45 µm polyethersulfone membrane) and avoid water loss during storage as this significantly decreases the transformation efficiency. Store at room temperature
  5. CSM-minimal media
    0.7 g/L YNB (without ammonium sulphate, without potassium)
    5 g/L ammonium sulphate
    1 g/L potassium dihydrogen orthophosphate
    20 g/L glucose
    0.55 g/L of CSM-dropout mix, adjust pH to 6.0 with fresh prepared KOH
    Add 20 g/L agar if solid media is needed. The YNB and CSM-dropout-mixes contained all amino acids apart from the desired selective one(s). Sterilize by autoclaving and store at 4 °C
  6. Reagents for auxotrophy selection
    Prepared by dissolving the following each chemical in 100 ml of water, sterilized by filtration, and stored in darkness at 4 °C:
    ADE: 0.4 g of adenine sulphate (add 5 ml per liter media)
    URA: 0.4 g of uracil (add 5 ml per liter media)
    LEU: 2.0 g of L-leucine (add 5 ml per liter media)
    TRP: 0.4 g of L-tryptophane (add 5 ml per liter media)
    HIS: 0.4 g of L-histidine (add 5 ml per liter media)
    MET: 1.5 g of L-methionine (equals a 0.1 M stock solution, add appropriate amount to obtain 0.5, 5, 50, and 500 μM final concentrations)
  7. CSM-auxotrophy selection media
    The CSM-auxotrophy selection media are prepared by adding reagents for auxotrophy selection (see Recipe 6) into the CSM-minimal media after autoclaving and cooling down to around 50 °C as shown below (Table 3):

    Table 3. The SC-auxotrophy selection media used for mbSUS assay
    Media
    Reagents for auxotrophy selection
    Comment
    CSM-TUM media
    ADE, HIS, LEU
    Transformation of Nub-clones in THY.AP5.
    CSM-LM media
    ADE, HIS, TRP, URA
    Transformation of Cub-clones in THY.AP4.
    CSM-LTUM media
    ADE, HIS
    Yeast mating check
    CSM-LTUMAH media with Met
    MET
    Yeast growth check; different methionine
    concentrations can help to increase the signal-to-noise ratio

  8. ‘Lyse & Load’ buffer
    50 mM Tris-HCl (pH 6.8)
    4% SDS
    8 M urea
    30% glycerol
    0.1 M DTT
    0.005% bromophenol blue
    Store at -20 °C
  9. 10% SDS-PAGE separation gel (50 ml)
    17 ml acrylamide (30%)
    12.5 ml 1.5 M Tris-HCl (pH 8.8)
    0.5 ml 10% SDS
    0.5 ml 10% ammonium persulfate
    0.05 ml TEMED
    Add ddH2O to 50 ml
  10. 5% SDS-PAGE stacking gel (50 ml)
    8.3 ml acrylamide (30%)
    6.3 ml 1 M Tris-HCl (pH 6.8)
    0.5 ml 10% SDS
    0.5 ml 10% (NH4)2S2O8
    0.05 ml TEMED
    Add ddH2O to 50 ml
  11. Bjerrums buffer
    6.1 g/L Tris
    2.4 g/L glycine
    20% methanol
    Store at 4 °C
  12. Ponceau S solution
    2 g/L Ponceau S in 5 % acetic acid
    Store at room temperature
  13. TBS solution and TBS-Tween solution
    8 g/L NaCl
    0.2 g/L KCl 
    3 g/L Tris
    Adjust pH to 7.4 with HCl
    Store at room temperature
    Add Tween® 20 to a final concentration 0.1% to make TBS-Tween
  14. Running buffer
    3 g/L Tris
    14.4 g/L glycine
    1.5 g/L SDS
    Store at room temperature
  15. Blocking solution
    5% milk powder in TBS-Tween solution

Acknowledgments

This protocol was adapted and modified from a previous publication (Grefen et al., 2009). We thank Prof. Michael Blatt for his advice and support in preparing this article. We thank Dr Emily Larson for her useful suggestions. This work was supported by a Royal Thai Government Scholarship to WH (ST4718), a Chinese Scholarship Council studentship to BZ, and by funding from BBSRC grants BB/I024496/1, BB/K015893/1, BB/L001276/1, BB/M01133X/1, BB/M001601/1, and BB/L019205/1 to Prof. Blatt.

References

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  2. Grefen, C., Chen, Z., Honsbein, A., Donald, N., Hills, A. and Blatt, M. R. (2010). A novel motif essential for SNARE interaction with the K+ channel KC1 and channel gating in Arabidopsis. Plant Cell 22(9): 3076-3092.
  3. Grefen, C., Karnik, R., Larson, E., Lefoulon, C., Wang, Y., Waghmare, S., Zhang, B., Hills, A. and Blatt, M. R. (2015). A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion. Nat Plants 1: 15108.
  4. Grefen, C., Obrdlik, P. and Harter, K. (2009). The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS). Methods Mol Biol 479: 217-233.
  5. Marfori, M., Mynott, A., Ellis, J. J., Mehdi, A. M., Saunders, N. F., Curmi, P. M., Forwood, J. K., Boden, M. and Kobe, B. (2011). Molecular basis for specificity of nuclear import and prediction of nuclear localization. Biochim Biophys Acta 1813(9): 1562-1577.
  6. Obrdlik, P., El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent, V., Blaudez, D., Sanders, D., Revuelta, J.L., Boles, E., André, B., and Frommer, W.B. (2004). K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc Natl Acad Sci USA 101: 12242-12247.
  7. Zhang, B., Karnik, R., Waghmare, S., Donald, N. A. and Blatt, M. R. (2016). VAMP721 conformations unmask an extended motif for K+ channel binding and gating control. Plant Physiol.
  8. Zhang, B., Karnik, R., Wang, Y., Wallmeroth, N., Blatt, M. R. and Grefen, C. (2015). The Arabidopsis R-SNARE VAMP721 interacts with KAT1 and KC1 K+channels to moderate K+current at the plasma membrane. Plant Cell 27(6): 1697-1717.

简介

基于交配的分ubiquitin(mbSUS)测定是具有许多优点的经典酵母双杂交系统的替代方法。 mbSUS测定依赖于泛素降解途径作为蛋白质 - 蛋白质相互作用的传感器,并且它适用于测定细胞溶质或膜结合的全长蛋白质之间的相互作用。在这里,我们描述了已经用于检测K + 通道和SNARE蛋白之间的相互作用的mbSUS测定方案(Grefen等人,2010和2015; Zhang 等等,2015和2016)

背景 图1是mbSUS测定的概况。泛素部分被分成两半,N末端半突变(NubG)以避免重组。泛素部分(Cub)的C末端一半与转录报告基因复合物PLV(Protein A-LexA-VP16)连接。两种蛋白质(X和Y)分别与NubG和CubPLV融合产生蛋白质 - 蛋白质相互作用分析系统。转化后,酵母菌株THY.AP5含有NubG-X融合蛋白,而酵母菌株THY.AP4含有Y-CubPLV融合蛋白。在交配后,在二倍体酵母中,如果蛋白质X和Y彼此相互作用,则将重新组装功能性泛素,这导致PLV的切割。释放的转录蛋白复合物PLV可以开启报告基因(ADE2,HIS3),并允许酵母生长在选择性培养基上。


图1.泛素泛素系统 A.泛素分为两半,N末端野生型半胱氨酸(NubWt; NubI)和C末端半数(Cub)。 NubWt和Cub的重新组装导致通过泛素特异性蛋白酶(USP)释放转录报告复合物蛋白A-LexA-VP16(PLV)。 B.将N末端半胱氨酸13位的异亮氨酸突变为甘氨酸,产生抑制泛素重新组装的NubG蛋白。在二倍体酵母中,产生NubG-X和Y-CubPLV融合蛋白。如果X和Y不相互作用,则不会形成功能性泛素,并且酵母不能在选择性培养基上生长,如右上方的小图像所示。 C.如果X与Y相互作用,那么NubG和Cub可以重新组装成功能泛素,这导致PLV的释放。 PLV激活合成ADE和HIS的报道基因(ADE2,HIS3),其允许在选择性培养基上的酵母生长而没有这些化学物质,如右上方的小图像所示。

关键字:mbSUS, 酵母, 交配, 分裂泛素, 蛋白质 - 蛋白质相互作用, Gateway

材料和试剂

  1. 无菌牙签
  2. 圆底聚苯乙烯管,14毫升(购买无菌)(康宁,目录号:352051)
  3. 蓝色螺旋盖管,50ml(购买无菌; Cellstar)
  4. 螺旋盖管,2毫升(购买无菌; SARSTEDT)
  5. PCR管,0.2ml,平盖(通过高压灭菌灭菌)(STARLAB INTERNATIONAL,目录号:I1402-8100)
  6. 硝酸纤维素转移膜,BioTrace TM NT(Pall,目录号:66485)
  7. 1.5毫升螺旋盖管
  8. Minisart ®注射器过滤器,非致热原,0.2μm(Sartorius)
  9. 注射器滤芯w /0.45μm聚醚砜膜(VWR,目录号:28145-503)
  10. 印刷纸(VWR,印迹垫707)
  11. 培养皿55毫米(购买无菌)(Greiner Bio One International,目录号:628102)
  12. 120 x 120毫米(购自无菌)的方格培养皿(Greiner Bio One International,目录号:688102)
  13. 过滤嘴10,200,1000μl(生物圈 Plus,用于微量移液器,SARSTEDT)
  14. 在mbSUS测定中使用的酵母菌株(表1)
    酿酒酵母(Saccharomyces cerevisiae)
    表1.用于mbSUS测定的酵母菌株
    名称
    基因型
    功能
    参考
    THY.AP4

    ,leu2 - ,trp1 - br /> ura3 - ;
    lexA :: ADE2,
    lexA :: HIS3,lexA :: lacZ

    诱饵:携带Y-
    在向量中的CubPLV
    pMetYC-Dest
    (Obrdlik等人,2004)
    THY.AP5
    MAT
    URA3;
    ,leu2 - ,trp1 - >
    猎物:携带
    NubG-X在向量中
    pNX35-Dest或NubWt
    (Obrdlik等人,2004)

  15. 在mbSUS测定中使用的目的载体(表2)

    表2.用于mbSUS测定的目标载体
    矢量名称
    促销者
    网关站点
    功能
    参考
    pMetYC-Dest
    met25
    attR1,attR2
    融合蛋白与C-末端
    CubPLV作为诱饵;合成
    LEU在酵母中
    (Grefen等人,2009)
    pNX35-Dest
    ADH1
    attR1,attR2
    融合蛋白与N-末端
    NubG为猎物;合成
    酵母中的TRP
    (Grefen和Blatt,2012)

  16. 供体载体:pDONR207载体(Thermo Fisher Scientific)或其他供体载体含有attP1,attP2网关盒
  17. 网关®克隆酶:
    1. BP克隆酶 II酶混合物(Thermo Fisher Scientific,Invitrogen TM,目录号:11789020)
    2. LR Clonase ® II酶混合物(Thermo Fisher Scientific,Invitrogen TM,目录号:11791020)
  18. 蛋白梯(Pageruler Plus Prestained Protein Ladder,Thermo Fisher Scientific)
  19. Western印迹信号检测试剂盒(SuperSignal West Dura Chemiluminescent Substrate)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:37071)
  20. 抗体解决方案
    1. 主要:抗-HA(1:20,000,抗HA高亲和力大鼠单克隆抗体)(Roche Diagnostics,目录号:11867423001)或抗VP16(1:20,000,Rabbit中的抗VP16标签抗体)(Abcam,目录号号码:ab4808)
    2. 次级:抗兔HRP(1:20,000,山羊抗兔IgG-HRP)(Thermo Fisher Scientific,Invitrogen,目录号:G-21234)或抗大鼠HRP(1:2000 ,兔抗大鼠IgG H& L [HRP])(Abcam,目录号:ab6734)
  21. 甲硫氨酸
  22. 甘油(Fisher Scientific,目录号:10795711)
  23. 蛋白胨(Formedium TM ,目录号:PEP02)
  24. 葡萄糖(Fisher Scientific,目录号:10141520)
  25. 酵母提取物(Formedium TM ,目录号:YEA02)
  26. 氧化物琼脂(琼脂1号)(Oxoid,目录号:LP0011)
  27. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60377)
  28. 乙酸锂二水合物(Sigma-Aldrich,目录号:L4158)
  29. 去离子水
  30. 乙酸(Sigma-Aldrich,目录号:1.00063)
  31. 鲑鱼精DNA(ssDNA)(Sigma-Aldrich,目录号:31149)
  32. 聚乙二醇3350(Spectrum Chemical Manufacturing,目录号:Po125-500gm)
  33. YNB无硫酸铵,无钾(MP Biomedicals,目录号:114029622)
  34. 正磷酸二氢钾(Fisher Scientific,目录号:10783611)
  35. CSM-ADE-HIS-LEU-MET-TRP-URA(粉末)(MP Biomedicals,目录号:114560222)
  36. 琼脂
  37. 硫酸亚铁(Sigma-Aldrich,目录号:A3159)
  38. 尿嘧啶(Sigma-Aldrich,目录号:U1128)
  39. L-亮氨酸(Sigma-Aldrich,目录号:L8912)
  40. L-色氨酸(Sigma-Aldrich,目录号:T4196)
  41. L-组氨酸(Sigma-Aldrich,目录号:H3911)
  42. L-甲硫氨酸(Sigma-Aldrich,目录号:M5308)
  43. 十二烷基硫酸钠(SDS)(VWR,目录号:442444H)
  44. 尿素(Sigma-Aldrich,目录号:U5378)
  45. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43817或43815)
    注意:产品"43817"已经停产。
  46. 丙烯酰胺(Sigma-Aldrich,目录号:A8887)
  47. 溴酚蓝
  48. 过硫酸铵(Fisher Scientific,目录号:10020020)
  49. TEMED(Sigma-Aldrich,目录号:T9281)
  50. 硫酸铵(VWR,目录号:21333.296)
  51. Tris(Fisher Scientific,目录号:BP152-1)
  52. 甘氨酸(Fisher Scientific,目录号:10070150)
  53. 甲醇(Sigma-Aldrich,目录号:34860)
  54. Ponceau S(Sigma-Aldrich,目录号:P3504)
  55. 氯化钠(VWR,目录号:27810.295)
  56. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  57. (Sigma-Aldrich,目录号:P1379)
  58. 奶粉(Marvel,Iceland,UK)
  59. YPD媒体(见配方)
  60. LiAc溶液(参见食谱)
  61. ssDNA溶液(参见食谱)
  62. PEG溶液(参见食谱)
  63. CSM-minimal媒体(见配方)
  64. 营养缺陷型选择试剂(见食谱)
  65. SCM-营养缺陷型选择培养基(参见食谱)
  66. 'Lyse&加载'缓冲区(请参阅配方)
  67. 10%SDS-PAGE分离凝胶(参见食谱)
  68. 5%SDS-PAGE堆叠凝胶(见配方)
  69. Bjerrums缓冲区(见配方)
  70. Ponceau S解决方案(见配方)
  71. TBS溶液和TBS-Tween溶液(参见食谱)
  72. 运行缓冲区(见配方)
  73. 阻塞解决方案(见配方) 

设备

  1. 用于酵母在液体培养中生长的培养器振荡器(Sartorius,型号:BS1)
  2. 玻璃烧瓶500ml(通过高压灭菌灭菌)(Corning,PYREX ,目录号:4980-500)
  3. 高速冷冻微量离心机(Eppendorf,型号:5417 R)
  4. 用于大体积液体样品的冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM Legend TM RT)
  5. PCR机(VWR,PEQLAB,型号:peqSTAR 96 Universal Gradient)
  6. 移液器
  7. 用于固体培养基板上酵母生长的培养箱(Gallenkamp,型号:Ipr150)
  8. 超声波破碎机(MSE,型号:Soniprep 150 MSS150.CX,9.5 mm探针)
  9. 生物光度计(Eppendorf,型号:Biophotometer Plus 6132)
  10. SDS-PAGE凝胶电泳和印迹盒(Bio-Rad Laboratories,型号:Power PAC 1000和Mini-PROTEAN Tetra Cell)
  11. Shakes for Western印迹(Stuart,型号:Stuart STR9 Gyro摇杆)
  12. 半干印迹系统(VWR,PEQLAB,型号:PerfectBlue TM半干式印刷机)
  13. Western印迹成像平台(Vilber,型号:Fusion spectrum)
  14. Vortex(科学工业,型号:Vortex-Genie 2)
  15. 成像系统
  16. 高压釜

程序

  1. 分子生物学
    网关®技术(Thermo Fisher Scientific,Germany)用于按照制造商的说明制备用于酵母mbSUS测定的质粒。分别将正向和反向引物中独特的attB1和attB2位点(B1 5'-3':ggggacaagtttgtacaaaaaagcaggct; B2 5'-3':ggggaccactttgtacaagaaagctgggt)作为突出序列。为了将PCR产物与N末端标签融合在正确的阅读框架中,正向引物必须包含两个另外的核苷酸。根据目标载体,反向引物中包含终止密码子或载体本身。
    随后的PCR产物通过BP反应插入到pDONR207载体中,以产生通过DNA测序验证的进入克隆。网关目的地克隆,pNX35-Dest for prey(Grefen和Blatt,2012)和pMetYC-Dest for bait(Grefen等人,2009),是通过使用LR Clonase 根据制造商的说明书在LR反应中进行
  2. 酵母转化
    在无菌条件下进行酵母转化。为每次转化准备新鲜有效的酵母菌
    1. 通过无菌牙签从长期库存中挑选THY.AP4和AP5酵母,或从储备板中挑取一个酵母菌落(见注释7),并在14 ml无菌圆底管中接种5ml YPD,并在振荡器中孵育过夜200 rpm,28°C
    2. 在高压灭菌的500ml烧瓶中用5ml过夜培养物接种45ml YPD,并以200rpm,28℃振荡孵育3-5小时,直到OD 600至0.6-0.8。
    3. 通过在50ml蓝色螺旋盖管中离心(在2,000 xg,4℃下10分钟)收获细胞并弃去上清液。
    4. 用20ml无菌水洗涤细胞,再次离心,弃去上清液
    5. 用1.8ml的0.1M LiAc将细胞重新悬浮并转移到2ml Eppendorf管中,向下旋转(30℃,2,000xg,4℃),并弃去上清液。
    6. 加入适量的0.1M LiAc(将转化次数乘以20μl),并在室温下孵育30分钟。
    7. 同时,为每次转化制备具有10μlssDNA和5μl质粒DNA(目的克隆,> 100ng /μl)的无菌PCR管。
    8. 准备主混合物:每次转化,混合70μlPEG溶液,10μl1M LiAc和20μl感受态酵母(来自步骤B6)。
    9. 将主混合物与步骤B7的DNA混合物混合。
    10. 在PCR机中,在30℃孵育30分钟,然后用移液管和热休克细胞在43℃下轻轻混合30分钟。
    11. 将酵母以2,000 x g的速度旋转1分钟,然后将其从细胞中移出细胞,弃去上清液。
    12. 用100μl无菌水清洗细胞,旋转下来,弃去上清液。
    13. 将细胞重悬在50μl无菌水中并在适当的选择培养基上培养
      1. THY.AP4在CSM 固体培养基上携带诱饵载体板。
      2. THY.AP5在CSM 固体培养基上携带猎物载体板。
    14. 在28℃孵育48-72小时。每个板块至少有100个殖民地。

  3. mbSUS分析
    mbSUS测定在无菌条件下进行。
    1. 选择携带猎物构建体的转化THY.AP5酵母的5-15个菌落并接种在3ml CSM-TUM培养基中。在培养箱振荡器中以200 rpm,28°C生长过夜。
    2. 选择携带诱饵构建体的转化的THY.AP4酵母的5-15个菌落,并接种在3ml CSM-L1M培养基中。在培养箱振荡器中以200 rpm,28°C生长过夜。
    3. 从每个诱饵和猎物酵母培养物中保留750μl作为储存培养物储存(参见酵母储备制备注)。
    4. 通过在室温下以2,000×g离心收获酵母细胞1分钟,并将沉淀重悬于YPD培养基中(对于每个配对,应计算20μlYPD培养基)。
    5. 在灭菌的PCR管中混合20μl各种酵母(携带诱饵)和酵母(携带猎物),并立即将4μl的每种配对酵母放在YPD板上。在28°C孵育过夜。
    6. 使用无菌牙签将配对的酵母从YPD板转移到CSM 板上(图2A)。在28°C过夜生长。


      图2. mbSUS测定的实施例。A.用无菌牙签将酵母从YPD培养基转移到CSM 钠板上。 B.酵母被放在CSM -LTUMAH 板上,纸上有黑点。将表达KAT1-CubPLV的二倍体酵母作为饵料,将VAMP721,VAMP723和对照(阴性:NubG;阳性:NubWt)作为猎物的NubG-X融合物点样到不同的培养基上,如图所示。 CSM -LTUM 用于验证饵料和猎物载体的存在。使用不同浓度的甲硫氨酸(Met)与CSM -LTUMAH 相结合,以证明当K +/sup> channel-CubPLV表达被抑制时的相互作用。在每种情况下,二倍体酵母以1.0和0.1 OD 600滴下。 CSM 板的孵育时间为24小时,CSM-LTUMAH板的孵育时间为72小时。对于mbSUS使用的二倍体酵母的蛋白质印迹分析用用于VAMP融合物的商业HA抗体和K + 通道融合体的VP16抗体进行。 Ponceau S污渍被用作印迹/加载控制(右)
    7. 从CSM 板中挑取每个样品的配对酵母菌落,并接种于3ml液体CSM 培养基中,在培养箱振荡器中以200rpm,28 °C。
    8. 接种1ml的配对细胞到3ml新鲜的CSM 培养基中,并以200rpm,28℃生长4-6小时,用于随后的Western印迹分析。
    9. 确定来自步骤C7的酵母细胞的OD <600>。
    10. 通过在室温下2000分钟离心收获来自步骤C7的100μl剩余的细胞,并在无菌水中分别稀释至OD 600和1.0和0.1。 />
    11. 在具有不同甲硫氨酸浓度(0.5,5,50和500μM;图2B)的CSM -LTUM 和CSM 平板上涂抹4μl每种稀释液,生长他们在28°C。分别检查在CSM <! - SIPO - >板和CSM-L1TMAMAH板上生长1天和3天后的生长。图2C显示了mbSUS测定的一个实例,其表明VAMP721和KAT1K信号通道之间的相互作用,并且VAMP723和KAT1之间没有相互作用(Zhang等人,2015) 。含有NubWt作为猎物的二倍体酵母作为阳性对照,含有NubG作为牺牲品的酵母是阴性对照。在良好的mbSUS测定中,阴性对照样品在CSM-L1TMAMAH板上显示较弱或无酵母生长,而酵母菌在阳性对照样品中生长良好(图2C)。

  4. 酵母蛋白提取用于Western印迹分析
    1. 确定酵母培养物的OD 600
    2. 收获2毫升的文化,并重新悬挂在"Lyse&在每个样品的最终OD 600(小于100)处加载'缓冲液。 "Lyse&使用的负载缓冲液是这样计算的:例如:,1:10稀释的培养物测量得到0.5的OD 600,其相当于OD 600 为1 ml培养物。收获2ml培养物产生OD 600,然后将其重悬于0.1ml"Lyse&加载'缓冲区可以在样本中得到100的最终OD <600> 。
    3. 用超声波破碎机(振幅设定为10秒20秒,将样品保存在冰上)散发酵母。
    4. 在65℃孵育10分钟。
    5. 涡旋10秒,将溶胞产物全速旋转30秒,并将上清液转移到新鲜管中。然后样品可用于Western印迹分析。

  5. 蛋白质印迹分析
    1. 在SDS-PAGE凝胶上加载蛋白质梯和10-15μl各样品
    2. 在合适的条件下运行凝胶并将蛋白质转移到硝酸纤维素膜(例如通过半转移)。
    3. 在室温下,在封闭缓冲液中封闭膜60分钟
    4. 将膜转移到第一抗体溶液;在室温下孵育至少3小时或在4℃下孵育过夜。
    5. 用1x TBS-Tween洗涤3次,每次洗涤10分钟。
    6. 将膜转移到二次抗体溶液中;在室温下孵育至少3小时,或在4℃下孵育过夜。
    7. 用1x TBS-Tween洗涤3次,每次洗涤10分钟。
    8. 将化学发光底物的膜在黑暗中孵育5分钟
    9. 开发膜片并拍摄成像系统。
    10. 用蒸馏水多次洗涤膜以除去化学发光底物。
    11. 将Ponceau S溶液中的膜孵育5分钟
    12. 用蒸馏水洗涤膜几次,直到红色条纹清晰可见。
    13. 扫描Ponceau S图像作为印迹/加载控制。

笔记

  1. 当使用蛋白质融合物时,天然蛋白质与Y-CubPLV融合体的C-末端应位于胞质溶胶中,并且捕获的NubG-X融合体也应在细胞质溶胶中可用,以进行有效的相互作用。 Cub和Nub的重组然后导致PLV部分的切割,并允许该转录因子移动到细胞核中。
  2. 小于40kDa的大分子可被动地扩散通过核孔,一些较大的颗粒也能够通过孔的大直径(Marfori等人,2011)。因此,诱饵蛋白通常会锚定在膜上。否则可溶性诱饵融合物可能会进入核心,并激活报告基因,与任何相互作用和PLV切割无关。 Preys融合不需要被膜锚定。
  3. 诱饵载体pMetYC-Dest中的融合蛋白的表达在met25启动子的控制下,其抑制培养基中甲硫氨酸存在下的表达(Grefen等人,2009)。然而,我们对mbSUS测定的经验表明,不含外部甲硫氨酸的培养基上的酵母生长比添加5μM甲硫氨酸的培养基上的酵母慢。为了克服这个问题,建议在介质中加入0.5μM甲硫氨酸。通过降低诱饵表达,可以使用较高的浓度(高达1mM Met)来测试相互作用中的特异性
  4. 在酵母脱落之前,建议让培养基板干燥。允许板的表面在无菌条件下干燥30分钟。板中固体介质的干燥表面有助于保持酵母斑点。
  5. 当放酵母时,建议用纸上的黑点打印滴纸方案,将其贴在纸板下方,如图2B所示。
  6. 对于蛋白质印迹分析,我们建议酵母不被培养过夜。从之前的过夜生长中将3ml新鲜培养基加入到1ml酵母培养物中,并允许培养物在收获前再生长4-6小时。这种方法避免了可能导致降解蛋白质并导致差的蛋白质印迹结果的培养物中衰老酵母的可能性。
  7. 为了制备长期储存的酵母菌液,移取750微升新鲜的过夜酵母培养物(来自步骤C1或C2),并在1.5ml螺旋盖管中加入250μl无菌60%甘油,紧紧关闭盖子并在冷冻之前混合在-80℃。建议每6个月更新一次甘油储备,避免甘油储备的反复冻融循环。为了从甘油原料中回收酵母细胞,使用无菌牙签挑取冷冻的酵母细胞,滴落并铺在合适的选择性培养基固体板上,然后在28℃下孵育24-48小时。选择性培养基上的酵母菌落准备用于mbSUS测定,酵母菌落可以在4℃下储存长达1个月。为了制备用于短期储存的酵母菌液,移取5-10μl新鲜的过夜酵母培养物(来自步骤C1或C2)或从甘油储备中挑取冷冻的酵母细胞;并将细胞放入合适的选择性固体培养基上。培养酵母在28℃24-48小时,然后在4℃下储存长达1个月。

食谱

  1. YPD媒体
    2%蛋白胨
    2%葡萄糖
    1%酵母提取物
    2%琼脂
    用1N KOH调节pH至6.0 通过高压灭菌消毒并在4℃下储存
  2. LiAc溶液(1M或0.1M)
    将乙酸锂溶解在去离子水中。用乙酸将pH调节至7.5,通过过滤灭菌(Minisart 注射器过滤器)并在室温下储存
  3. ssDNA溶液
    将10 mg/ml ssDNA溶解在去离子水中,通过过滤灭菌(Minisart注射器过滤器),并在使用前在冰上冷却后煮沸5分钟。储存于-20°C
  4. PEG溶液
    将PEG 3350在去离子水中溶解至最终浓度为50%(w/v),通过过滤灭菌(0.45μm聚醚砜膜),并避免储存期间的水分损失,因为这显着降低了转化效率。在室温下存放
  5. CSM-minimal media
    0.7g/L YNB(不含硫酸铵,不含钾)
    5克/升硫酸铵
    1g/L正磷酸二氢钾
    20 g/L葡萄糖
    0.55g/L的CSM脱落混合物,用新鲜制备的KOH调节pH至6.0 如果需要固体培养基,则加入20 g/L琼脂。 YNB和CSM脱水混合物除了所需的选择性物质外还含有所有氨基酸。通过高压灭菌消毒并在4℃下储存
  6. 用于营养缺陷选择的试剂
    通过将以下每种化学品溶解在100ml水中制备,通过过滤灭菌,并在4℃的黑暗中储存:
    ADE:0.4克腺嘌呤硫酸盐(每升培养基加5ml)
    URA:0.4g尿嘧啶(每升培养基加5ml)
    LEU:2.0g L-亮氨酸(加入5ml /升培养基)
    TRP:0.4g L-色氨酸(加入5ml /升培养基)
    HIS:将0.4g L-组氨酸(加入5ml每升培养基)
    MET:1.5g L-甲硫氨酸(等于0.1M储备溶液,加入适量以得到0.5,5,50和500μM终浓度)
  7. CSM-营养缺陷选择媒体
    如下所示,通过将营养缺陷型选择(见方案6)的试剂加入到CSM基本培养基中并在高压灭菌后冷却至约50℃来制备CSM-营养缺陷型选择培养基(表3):

    表3.用于mbSUS测定的SC-营养缺陷型选择培养基

    媒体
    营养缺陷选择试剂
    评论
    CSM 媒体
    ADE,他的,LEU
    在THY.AP5中转化Nub克隆。
    CSM -LM 媒体
    ADE,HIS,TRP,URA
    在THY.AP4中转化Cub克隆。
    CSM -LTUM 媒体
    ADE,他的
    酵母交配检查
    CSM -LTUMAH 媒体与Met
    MET
    酵母生长检查;不同的蛋氨酸
    浓度有助于提高信噪比

  8. 'Lyse&加载缓冲区
    50mM Tris-HCl(pH6.8)
    4%SDS
    8 M尿素
    30%甘油
    0.1 M DTT
    0.005%溴酚蓝
    储存于-20°C
  9. 10%SDS-PAGE分离凝胶(50ml) 17ml丙烯酰胺(30%)
    12.5ml 1.5M Tris-HCl(pH8.8)
    0.5 ml 10%SDS
    0.5ml 10%过硫酸铵
    0.05 ml TEMED
    将ddH 2 O添加到50 ml
  10. 5%SDS-PAGE堆叠凝胶(50ml)
    8.3毫升丙烯酰胺(30%)
    6.3ml 1M Tris-HCl(pH6.8)
    0.5 ml 10%SDS
    0.5ml 10%(NH 4)2 N 2 S 2 O 8// 0.05 ml TEMED
    将ddH 2 O添加到50 ml
  11. Bjerrums缓冲区
    6.1 g/L Tris
    2.4 g/L甘氨酸
    20%甲醇
    储存于4°C
  12. Ponceau S解决方案
    2g/L Ponceau S在5%乙酸中 在室温下存放
  13. TBS溶液和TBS-Tween溶液
    8克/升NaCl
    0.2克/升KCl
    3克/升Tris
    用HCl调节pH至7.4
    在室温下存放
    添加吐温® 20至终浓度0.1%,使TBS-Tween
  14. 运行缓冲区
    3克/升Tris
    14.4g/L甘氨酸
    1.5克/升SDS
    在室温下存放
  15. 阻塞解决方案
    TBS-Tween溶液中的5%奶粉

致谢

该协议从先前的出版物(Grefen等人,2009)进行了修改和修改。我们感谢Michael Blatt教授在准备这篇文章时的建议和支持。我们感谢Emily Larson博士的有用建议。这项工作得到泰国皇家政府奖学金的支持,WH(ST4718),中国奖学金委员会对BZ的奖学金,以及BBSRC拨款BB/I024496/1,BB/K015893/1,BB/L001276/1,BB/Prof. Blatt的M01133X/1,BB/M001601/1和BB/L019205/1。

参考

  1. Grefen,C.和Blatt,MR(2012)。钙调磷酸酶B样蛋白与K + 通道AKT1的丝氨酸苏氨酸激酶CIPK23独立相互作用从一个trois获得的经验教训。植物生理学159(3):915-919。
  2. Grefen,C.,Chen,Z.,Honsbein,A.,Donald,N.,Hills,A.and Blatt,MR(2010)。  与K + 通道KC1和拟南芥中的通道门控的SNARE相互作用所必需的新颖主题, em>。植物细胞 22(9):3076-3092。
  3. Grefen,C.,Karnik,R.,Larson,E.,Lefoulon,C.,Wang,Y.,Waghmare,S.,Zhang,B.,Hills,A.and Blatt,MR(2015)一个class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27250541"target ="_ blank">一个囊泡贩运蛋白质指挥官Kv通道电压传感器用于电压依赖性分泌植物 1:15108.
  4. Grefen,C.,Obrdlik,P.和Harter,K。(2009)。通过基于交配的分裂泛素系统(mbSUS)测定蛋白质 - 蛋白质相互作用。方法Mol Biol 479:217-233。
  5. Marfori,M.,Mynott,A.,Ellis,JJ,Mehdi,AM,Saunders,NF,Curmi,PM,Forwood,JK,Boden,M。和Kobe,B。(2011)。核导入和核定位预测的特异性的分子基础 Biochim Biophys Acta 1813(9):1562-1577。
  6. Obrdlik,P.,El-Bakkoury,M.,Hamacher,T.,Cappellaro,C.,Vilarino,C.,Fleischer,C.,Ellerbrok,H.,Kamuzinzi,R.,Ledent,V.,Blaudez,D 。,Sanders,D.,Revuelta,JL,Boles,E.,André,B.和Frommer,WB (2004)。 K + 。Proc Natl Acad Sci USA 101:12242-12247。
  7. Zhang,B.,Karnik,R.,Waghmare,S.,Donald,NA and Blatt,MR(2016)。  VAMP721构象取消掩蔽K + 通道结合和门控控制的扩展基序。植物生理学。
  8. Zhang,B.,Karnik,R.,Wang,Y.,Wallmeroth,N.,Blatt,MR and Grefen,C.(2015)。  拟南芥 R-SNARE VAMP721与KAT1和KC1 K + 通道相互作用以适应K + 电浆在质膜上。植物细胞 27(6):1697-1717。
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引用:Horaruang, W. and Zhang, B. (2017). Mating Based Split-ubiquitin Assay for Detection of Protein Interactions. Bio-protocol 7(9): e2258. DOI: 10.21769/BioProtoc.2258.
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