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CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca2+/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms.

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CytoTrap Two-Hybrid Screening Assay
CytoTrap双杂交筛选系统

植物科学 > 植物生物化学 > 蛋白质 > 相互作用
作者: Lei Zhang
Lei ZhangAffiliation 1: Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, USA
Affiliation 2: Department of Horticulture, Washington State University, Pullman, USA
Present address: Department of Plant Pathology, Washington State University, Pullman, USA
Bio-protocol author page: a1842
Liqun Du
Liqun DuAffiliation 1: Department of Horticulture, Washington State University, Pullman, USA
Affiliation 2: College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China
Bio-protocol author page: a1843
 and B. W. Poovaiah
B. W. PoovaiahAffiliation 1: Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, USA
Affiliation 2: Department of Horticulture, Washington State University, Pullman, USA
For correspondence: poovaiah@wsu.edu
Bio-protocol author page: a1844
Vol 4, Iss 23, 12/5/2014, 3487 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1310

[Abstract] CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca2+/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms.
Keywords: Cytotrap system(CytoTrap系统), Yeast two-hybrid screen(酵母双杂交筛选), Protein interaction(蛋白质相互作用)

[Abstract]

Materials and Reagents

  1. XL10-Gold Kanr Ultracompetent cells (Agilent, catalog number: 200317 )
  2. RNeasy plant mini kit (QIAGEN, catalog number: 74904 )
  3. Oligotex mRNA mini kit (QIAGEN, catalog number: 70022 )
  4. CytoTrap two-hybrid system (Agilent, catalog number: 200444 )
  5. YeastmakerTM yeast transformation system 2 (Takara Bio Company, Clontech, catalog number: 630439 )
  6. AccuScript (Agilent, catalog number: 200820 )
  7. RNase H (New England Biolabs, catalog number: M0297S )
  8. DNA polymerase I (New England Biolabs, catalog number: M0209S )
  9. UltraPureTM phenol: chloroform: isoamyl alcohol (25:24:1, v/v) (Life Technologies, catalog number: 15593-031 )
  10. T4 ligase (New England Biolabs, catalog number: M0202S )
  11. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  12. Xho I (New England Biolabs, catalog number: R0146M )
  13. Acid-washed glass beads (Sigma-Aldrich, catalog number: G8772 )
  14. TritonTM X-100 (Thermo Fisher Scientific, catalog number: BP151-100 )
  15. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: BP166-100 )
  16. Sodium chloride (NaCl) (J.T.Baker®, catalog nuber: JT3628-1 )
  17. Tris (J.T.Baker®, catalog number: JT4109-1 )
  18. EDTA (J.T.Baker®, catalog number: JT8993-1 )
  19. Yeast extract (Thermo Fisher Scientific, catalog number: BP9727-500 )
  20. Peptone (Thermo Fisher Scientific, catalog number: BP9725-500 )
  21. Dextrose (J.T.Baker®, catalog number: JT1919-1 )
  22. Adenine sulfate (Sigma-Aldrich, catalog number: A2545 )
  23. Yeast nitrogen base without amino acids (BD, catalog number: 291940 )
  24. DO supplement -His/-Leu/-Trp/-Ura (Takara Bio Company, Clontech, catalog number: 630425 )
  25. Histidine (Sigma-Aldrich, catalog number: H6034 )
  26. Tryptophan (Sigma-Aldrich, catalog number: T0254 )
  27. Galactose (Sigma-Aldrich, catalog number: G0750 )
  28. Raffinose (Sigma-Aldrich, catalog number: R0250 )
  29. Yeast lysis solution (see Recipes)
  30. YPDA/YPAD (see Recipes)
  31. SC/-LU (glucose) (see Recipes)
  32. SC/-LU (galactose) (see Recipes)
  33. 10x STE buffer (see Recipes)

Equipment

  1. Replica plating mold (Sunrise Science Products, catalog number: 3005-002 )
  2. RNase-free microcentrifuge tube (BioExpress, catalog numer: C-3262-2 )
  3. Microcentrifuge (Eppendorf, catalog number: 5424 )
  4. Shaker (New Brunswick Scientific, catalog number: M1299-0092 )
  5. Incubator (VWR International, catalog number: 414005-128 )
  6. 150-mm plate (VWR International, catalog number: 25384-326 )
  7. PCR thermocycler (Eppendorf, catalog number: 6321 000.515 )

Procedure

  1. Constructing and testing bait-hSos fusion protein
    1. DNA encoding bait protein is cloned into the pSos vector (plasmid map: http://www.chem-agilent.com/pdf/strata/217438.pdf) by the conventional enzyme-ligation method. The plasmid construct is confirmed by DNA sequencing to ensure the correct reading frame between hSos and bait.
    2. hSos-bait should be tested for auto-activation or interact with the myristylation signal prior to the CytoTrap two-hybrid screen (Aronheim et al., 1997).
      1. The pSos bait plasmid needs to be co-transformed into yeast host cell cdc25H with negative control either pMyr or pMyr-Lamin C using YeastmakerTM Yeast Transformation System 2.
      2.  All the yeast culture procedures need to be conducted at room temperature (22-25 °C). After co-transformation, yeast cells are spread on synthetic media plate without leucine and uracil (SC/-LU) containing glucose, and incubate at room temperature for 4-5 days to allow yeast colonies to grow.
      3. Six independent colonies are picked and placed on SC/-LU medium plate containing galactose, and the plates are kept at room temperature for 1 h, and then transferred to 36 °C for 3 to 6 days for yeast growth.
        1. If the yeast cells co-transformed with the tested bait plasmid and empty pMyr or pMyr-Lamin C are able to grow at 36 °C, then it will be considered unsuitable for the CytoTrap two-hybrid system.
        2. Only bait plasmids that do not lead to growth of host yeast cells at 36 °C in the co-transformation test could be suitable to screen for interaction partners using the CytoTrap two-hybrid system.

  2. Generating a CytoTrap two-hybrid cDNA library
    CytoTrap two-hybrid cDNA libraries can be constructed using mRNA from various organisms. Here we use the mRNA from the model plant Arabidopsis as an example. Briefly, mRNA is extracted and purified from leaf tissue of wild-type Arabidopsis plant (Col-0), then first-strand cDNA is synthesized using a hybrid oligo (dT) linker-primer that contains an XhoI restriction site and a high fidelity reverse transcriptase. 5-methyl dCTP is used in place of regular dCTP during the synthesis of first strand cDNA, and the methylated XhoI sites in the resulting cDNA will be resilient to XhoI digestion. The second-strand cDNA is synthesized using DNA polymerase I. An adapter containing a cohesive EcoRI end and a blunt end (see sequence in Note 1) is linked to the polished double stranded cDNA, and then the cDNA was digested with XhoI. The resulting cDNA containing EcoRI at 5’ end and XhoI at 3’ end is ligated into pMyr vector pre-cut by EcoRI and XhoI, so the cDNA is inserted in a sense expressional orientation driven by the Gal1 promoter (PGAL1). The cDNA library carried in pMyr plasmid (plasmid map: http://www.chem-agilent.com/pdf/strata/217438.pdf) is then transformed into high efficiency E. coli XL10-Gold Kan cells for propagation and storage. The detailed procedure is described below.
    cDNA synthesis:
    1. mRNA purification. Leaf samples (~2 g) from 4-week-old Arabidopsis Col-0 plants are harvested, and total RNA is extracted from the leaf tissues using RNeasy Plant Mini Kit. Poly (A) mRNA is then purified from 1 mg of total RNA using Oligotex mRNA Mini Kit for cDNA synthesis (Zhang et al., 2014).
    2. First-strand cDNA synthesis is carried out using high fidelity reverse transcriptase AccuScript.
      1. Add the following reagents to an RNase-free microcentrifuge tube:
        10x first-strand buffer: 5 μl
        First-strand methyl nucleotide mixture: 3 μl
        Linker-primer (1.4 μg/μl): 2 μl
        RNase Block Ribonuclease Inhibitor (40 U/μl): 1 μl
        mRNA: Equal to 5 μg
        DEPC-treated water: Bring volume to 50 μl
      2. Incubate the reaction at room temperature for 10 min to allow primer to anneal to template.
      3. Add 3 μl of AccuScript reverse transcriptase to the reaction and incubate the reaction at 42 °C for 2 h.
    3. Second-strand cDNA synthesis.
      1. Put the first-strand cDNA reaction tube on ice, and add the following reagents to the tube:
        10x second-strand buffer: 20 μl
        Second-strand dNTP mixture: 6 μl
        Sterile ddH2O: 139 μl
        RNase H (5 U/μl): 1 μl
        DNA polymerase I (9.0 U/μl): 11 μl
      2. Mix the reaction gently and spin down the reaction.
      3. Incubate at 16 °C for 3 h, and then put the tube on ice. The resulting double-strand cDNA (ds cDNA) should be ready for adapter ligation.
    4. Blunting cDNA termini and ligating the EcoR I adapter.
      1. Add the following reagents to second-strand cDNA synthesis, and incubate at 72 °C for 30 min.
        10 mM dNTP mix: 4 μl
        Phusion DNA polymerase (NEB): 4 μl
      2. Add 200 μl UltraPureTM phenol: chloroform: isoamyl alcohol (25:24:1) to the reaction and vortex to mix. Spin down the reaction at 14,000 x g at room temperature for 5 min, and then transfer the upper layer (~ 200 μl) to a new tube.
      3. Add 200 μl chloroform and vortex. Spin down the reaction at 14,000 x g at room temperature for 5 min, and then transfer the upper layer (~ 200 μl) to a new tube.
      4. Add the following reagents to precipitate cDNA, vortex and keep the reaction at -20 °C overnight.
        3 M sodium acetate: 20 μl
        100% (v/v) ethanol: 400 μl
      5. Spin down the cDNA at 14,000 x g for 60 min at 4 °C.
      6. Remove the supernatant carefully and wash the pellet using 70% ethanol (v/v). Spin down the pellet at 14,000 x g for 2 min at room temperature. Remove the ethanol and dry the pellet at room temperature.
      7. Resuspend the pellet in 8 ul EcoRI adapters and incubate at 4 °C for 1 h to ensure the complete dissolve of cDNA.
      8. Add the following reagents to the tube with cDNA and EcoRI adapters to bring volume to 10 μl, and incubate the reaction at 16 °C for overnight.
        10x T4 ligase buffer (NEB): 1 μl
        T4 ligase (NEB): 1 μl
      9. Incubate the tube at 70 °C for 30 min to terminate the reaction.
    5. Phosphorylating the EcoRI end.
      1. Cool down the reaction at room temperature for 10 min.
      2. Add the following reagents to the tube, and incubate at 37 °C for 1 h.
        10x reaction buffer (NEB): 1 μl
        Sterile ddH2O: 8 μl
        T4 polynucleotide kinase (NEB): 2 μl
      3. Incubate the tube at 70 °C for 30 min to terminate the reaction.
      4. Cool the reaction at room temperature for 10 min, and then add the following reagents to the reaction, incubate at 37 °C for 3 h.
        10x reaction buffer (NEB): 5 μl
        Sterile ddH2O: 22 μl
        Xho I (NEB): 3 μl
      5. Precipitate the cDNA by adding the following reagents to the tube, and incubate at -20 °C overnight.
        10x STE buffer: 5 μl
        100% (v/v) ethanol: 125 μl
      6. Spin down the reaction at 14,000 x g for 60 min at 4 °C. Remove the supernatant and dry the pellet at room temperature. Dissolve the pellet in 100 μl sterile ddH2O.
    6. Purify ds cDNA with CHROMA SPINTM+TE-400 columns. (Procedures are performed at room temperature if not specified.)
      1. Resuspend the gel matrix by invert the column several times. Remove the top cap and take away the break-away end at the bottom. Place the column in a 2-ml collection tube.
      2. Centrifuge at 700 x g for 5 min, and then remove the collection tube and buffer.
      3. Put the column to a new collection tube and add 100 μl cDNA sample to the center of top surface of gel matrix.
      4. Centrifuge at 700 x g for 5 min, collect the elution solution containing cDNA (size >200 bp).
      5. Precipitate cDNA by adding the following reagents to the eluted solution, and incubate at -20 °C freezer for overnight.
        3 M sodium acetate (pH5.2): 10 μl
        Ice-cold 100% ethanol: 250 μl
      6. Centrifuge at 14,000 x g for 30 min at 4 °C. Discard the supernatant, and add 360 μl 70% ethanol, inverted several times, and then centrifuge at 14,000 x g for 5 min at 4 °C. Remove the supernatant and dry the pellet at room temperature. Resuspend the cDNA in 5 μl sterile ddH2O.
    7. Ligating cDNA into pMyr vector and transform into E. coli.
      1. Set up T4 ligation reaction by adding the following reagents, incubate overnight at 16 °C.
        10x ligase buffer (NEB): 2.5 μl
        Resuspended cDNA (1 μg): X μl
        pMyr vector (precut by EcoRI/XhoI, 500 ng): 5 μl
        T4 DNA ligase (NEB): 2.5 μl
        ddH2O: Bring volume to 25 μl
      2. Transform all the ligation reaction to XL10-Gold Kanr Ultracompetent cells. Use 2 μl ligation reaction for each transformation according to the manufacturer’s instruction.
      3. Plate the transformation on 150-mm LB plates containing chloramphenicol (25 μg/ml), about 50-100 plates in total. Spread appropriate volume of transformation, so that each plate may have 30,000 colonies. Incubate at 37 °C overnight to allow colonies to grow. The total number of colonies should be 3 million.
      4. Add 5 ml LB medium to each plate to scrape the bacteria with a spreader, and wash each plate using additional 1 ml LB medium. Pool all the bacteria in one beaker and mix gently and well. Aliquot 10 ml bacteria culture in 15-ml centrifuge tubes and stored at -80 °C. Purify plasmid from each 10 ml aliquot for CytoTrap two-hybrid screen.

  3. Screening a CytoTrap two-hybrid cDNA library
    The bait and cDNA library plasmids are co-transformed into temperature sensitive host strain cdc25H. The transformation reaction is spread on SC/-LU plates containing glucose at room temperature to allow positive transformants to grow, and then the transformants are replica plated to SC/-LU plates containing galactose and incubated at 36 °C to select clones which expressing the bait and a potential interaction partner genes. The positive clones are further analyzed to confirm the bait-specific protein interaction. The flowchart of library screening is shown in Figure 1.
    1. Co-transformation of bait and cDNA library plasmids into host strain cdc25H.
      This step is conducted as suggested by YeastmakerTM Yeast Transformation System 2 manual except that all the yeast culture steps must be conducted at room temperature unless otherwise specified to reduce rate of revert mutation of cdc25H. The transformation reactions are spread on SC/-LU plates (150-mm) containing glucose and kept at room temperature for 3 to 4 days until colonies grow to 2-3 mm in diameter. It is ideal to have ~3,000 colonies on each plate, and it is necessary to screen at least 1 million colonies.
    2. Replica plate the colonies grown up to SC/-LU plates (150-mm) containing galactose, and leave the plates at room temperature for one hour before moving to the selective temperature of 36 °C for incubation of 3 to 6 days. Colonies containing putative interacting partners and reverting mutants will grow up and it is necessary to pick up the colonies when they are 2-3 mm in diameter during selection, and not wait to pick up at the end of selection.
    3. Pick up colonies grown up on SC/-LU plates containing galactose at 36 °C, and dissolve the colonies in 25 μl sterile dH2O. Dot 5 μl of yeast suspension to four plates: Two SC/-LU containing glucose and two SC/-LU containing galactose. Incubate one SC/-LU plate containing glucose and one SC/LU plate containing galactose at room temperature, and the other two plates at 36 °C.
      Notes:
      1. The number of plates should be scaled up if there are more yeast colonies.
      2. As shown in Figure 1, yeast colonies that only grow on SC/-LU containing galactose, but not on SC/LU containing glucose at 36 °C are kept as primary positive candidates for further analysis.
      3. The yeast colonies that grow on both galactose and glucose plates at 36 °C are temperature revert mutants, and their growth at selective temperature is caused by revert mutation of cdc25H mutant gene back to wild-type gene, hence their growth at 36 °C is not galactose-dependent.
      4. A large percentage of false positives will be the temperature revert mutants and can be easily identified by testing galactose-dependent growth at 36 °C.


        Figure 1. CytoTrap two-hybrid library screening procedure. A pSos-bait is co-transformed into yeast strain cdc25H with a cDNA library in pMyr. Positive transformants are selected on SC/-LU (glucose) plates at room temperature. Colonies are then replica plated on SC/-LU (galactose) plates and incubated at 36 °C for 3 to 6 days. Colonies grown up at 36 °C are further tested for galactose- and bait-specific growth to identify the true interaction clones.

  4. Analyze positive interaction candidates
    The primary positive candidates from library screen need to be further tested to see whether the interaction is bait-specific and nature of the prey genes need to be identified for functional analysis.
    1. Isolate prey plasmids from positive candidates.
      1. Inoculate positive yeast candidates in 2 ml of liquid SC/-LU containing glucose and shake at 30 °C for 2 days.
      2. Transfer 1.5 ml yeast culture to a 1.7-ml centrifuge tubes, and spin down the cells at 14, 000 x g for 1 min. Remove the supernatant and add 200 μl yeast lysis solution, resuspend cells by vortexing.
      3. Add 200 μl UltraPureTM phenol: chloroform: isoamyl alcohol (25:24:1, v/v) and 0.3 g of acid-washed glass beads, vortex vigorously for 2 min for each sample.
      4. Centrifuge at 14,000 x g for 5 min at room temperature, and transfer the top layer into a new microcentrifuge tube. Add the following reagents to each tube, and keep the tubes at -20 °C for 30 min.
        3 M sodium acetate (pH 5.2): 20 μl
        Ice-cold 100% ethanol: 500 μl
      5. Spin down the pellet at 14,000 x g for 10 min at 4 °C. Remove the supernatant, and wash the pellet with 70% ethanol. Spin down the pellet at 14,000 x g for 5 min at room temperature. Remove the supernatant and dry the pellet at room temperature.
      6. Resuspend the pellet in 50 μl sterile ddH2O, and then use 1-2 μl of yeast plasmid DNA to transform 10-beta Electrocompetent E. coli competent cells. Spread the transformation reaction on LB/chloramphenicol agar plates. Put at 37 °C for overnight.
      7. Pick up single colonies and inoculate in LB/chloramphenicol liquid medium at 37 °C for overnight. Purify the plasmid for further analysis.
    2. Yeast co-transformation to test bait-specific protein-protein interaction
      1. Transform the purified candidate prey plasmids with either pSos-bait or pSos empty vector into host strain cdc25H.
      2. Inoculate the transformants on SC/-LU containing glucose at room temperature for 4 days. Pick up three individual colonies and transfer to SC/-LU containing glucose and SC/-LU containing galactose plates, and incubate at 36 °C for 3 to 6 days.
      3. Only prey plasmids that show specifically interact with pSos-bait, not with pSos empty vector are kept for sequence analysis.
      4. The identity of cDNA insert is determined by DNA sequencing.

Notes

  1. Sequence of adaptor containing a cohesive EcoR I end and a blunt end:
    5´-OH-AATTCGGCACGAGG-3´
    3´-GCCGTGCTCCp-5´

Recipes

  1. Yeast lysis solution (100 ml)
    2 ml Triton® X-100
    1 g SDS
    0.58 g NaCl
    1 ml of 1 M Tris-HCl (pH 8.0)
    200 μl of 0.5 M EDTA (pH 8.0)
    Bring volume to 100 ml using sterile dH2O
    Stored at room temperature
  2. YPDA/YPAD (1 L)
    10 g yeast extract
    20 g Bacto® peptone
    20 g dextrose
    40 mg adenine sulfate
    Bring volume to 1 L using sterile dH2O, and adjust to pH 5.7-5.8
    If preparing plates, add 15 g agar
    Autoclave at 121 °C for 20 min
  3. SC/-LU (glucose, 1 L)
    6.7 g yeast nitrogen base without amino acids
    20 g dextrose
    0.6 g DO Supplement -His/-Leu/-Trp/-Ura
    0.02 g histidine
    0.05 g tryptophan
    Bring volume to 1 L using sterile dH2O, and adjust to pH 5.7-5.8
    If preparing plates, add 15 g agar
    Autoclave at 121 °C for 20 min
  4. SC/-LU (galactose, 1 L)
    6.7 g yeast nitrogen base without amino acids
    20 g of galactose
    10 g of raffinose
    0.6 g DO supplement -His/-Leu/-Trp/-Ura
    0.02 g histidine
    0.05 g tryptophan
    Bring volume to 1 L using sterile dH2O, and adjust to pH 5.7-5.8
    If preparing plates, add 15 g agar
    Autoclave at 121 °C for 20 min
  5. 10x STE buffer (1 L)
    58.44 g NaCl
    200 ml of 1 M Tris-HCl (pH 7.5)
    200 ml of 0.5 M EDTA
    Bring volume to 1 L using sterile dH2O

Acknowledgments

The research was supported by National Science Foundation grant 1021344 to B.W. Poovaiah, and the National Science Foundation of China grant U1130304 to Liqun Du. We thank Lorie Mochel and Ade Snider at Washington State University for their help in preparing the manuscript.

References

  1. Aronheim, A., Zandi, E., Hennemann, H., Elledge, S. J. and Karin, M. (1997). Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol 17(6): 3094-3102.
  2. Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D., Margolis, B. and Schlessinger, J. (1993). Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363(6424): 85-88.
  3. Zhang, L., Du, L., Shen, C., Yang, Y. and Poovaiah, B. W. (2014). Regulation of plant immunity through ubiquitin-mediated modulation of Ca(2+) -calmodulin-AtSR1/CAMTA3 signaling. Plant J 78(2): 269-281.

材料和试剂

  1. XL10-Gold Kanr Ultracompetent细胞(Agilent,目录号:200317)
  2. RNeasy植物迷你试剂盒(QIAGEN,目录号:74904)
  3. Oligotex mRNA小试剂盒(QIAGEN,目录号:70022)
  4. CytoTrap双杂交系统(Agilent,目录号:200444)
  5. 酵母转化系统2(Takara Bio Company,Clontech,目录号:630439)。
  6. AccuScript(Agilent,目录号:200820)
  7. RNase H(New England Biolabs,目录号:M0297S)
  8. DNA聚合酶I(New England Biolabs,目录号:M0209S)
  9. UltraPure TM酚:氯仿:异戊醇(25:24:1,v/v)(Life Technologies,目录号:15593-031)
  10. T4连接酶(New England Biolabs,目录号:M0202S)
  11. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  12. Xho I(New England Biolabs,目录号:R0146M)
  13. 酸洗玻璃珠(Sigma-Aldrich,目录号:G8772)
  14. TritonX-100(Thermo Fisher Scientific,目录号:BP151-100)
  15. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientific,目录号:BP166-100)
  16. 氯化钠(NaCl)(J.T.Baker ,目录编号:JT3628-1)
  17. Tris(J.T.Baker ,目录号:JT4109-1)
  18. EDTA(J.T.Baker ,目录号:JT8993-1)
  19. 酵母提取物(Thermo Fisher Scientific,目录号:BP9727-500)
  20. 蛋白胨(Thermo Fisher Scientific,目录号:BP9725-500)
  21. 葡萄糖(J.T.Baker ,目录号:JT1919-1)
  22. 硫酸腺嘌呤(Sigma-Aldrich,目录号:A2545)
  23. 无氨基酸的酵母氮源(BD,目录号:291940)
  24. DO补充-His/-Leu/-Trp/-Ura(Takara Bio Company,Clontech,目录号:630425)
  25. 组氨酸(Sigma-Aldrich,目录号:H6034)
  26. 色氨酸(Sigma-Aldrich,目录号:T0254)
  27. 半乳糖(Sigma-Aldrich,目录号:G0750)
  28. 棉子糖(Sigma-Aldrich,目录号:R0250)
  29. 酵母裂解液(见配方)
  30. YPDA/YPAD(请参阅配方)
  31. SC/-LU(葡萄糖)(参见配方)
  32. SC/-LU(半乳糖)(参见配方)
  33. 10x STE缓冲液(参见配方)

设备

  1. 复制电镀模具(Sunrise Science Products,目录号:3005-002)
  2. 无RNase的微量离心管(BioExpress,目录号:C-3262-2)
  3. 微量离心机(Eppendorf,目录号:5424)
  4. Shaker(New Brunswick Scientific,目录号:M1299-0092)
  5. 孵化器(VWR International,目录号:414005-128)
  6. 150-mm板(VWR International,目录号:25384-326)
  7. PCR热循环仪(Eppendorf,目录号:6321 000.515)

程序

  1. 构建和测试bait-hSos融合蛋白
    1. 将编码诱饵蛋白的DNA克隆到pSos载体中(质粒图: http://www .chem-agilent.com/pdf/strata/217438.pdf ) 酶连接法。 通过DNA确认质粒构建体 测序以确保hSos和诱饵之间的正确阅读框。
    2. hSos诱饵应测试自动激活或与之交互 在CytoTrap双杂交筛选之前的肉豆蔻酸化信号(Aronheim等人,1997)。
      1. pSos诱饵质粒需要共转化 进入酵母宿主细胞cdc25H与阴性对照pMyr或 pMyr-Lamin C,使用Yeastmaker TM 酵母转化系统2.
      2.  所有酵母培养程序需要在室内进行 温度(22-25℃)。 共转化后,酵母细胞被扩散 在不含亮氨酸和尿嘧啶(SC/-LU)的合成培养基平板上 葡萄糖,并在室温下孵育4-5天以允许酵母 殖民地生长。
      3. 挑取并放置六个独立的菌落   在含有半乳糖的SC/-LU培养基平板上,并保持平板   室温下1小时,然后转移至36℃3至6天   用于酵母生长
        1. 如果酵母细胞与 测试诱饵质粒和空pMyr或pMyr-Lamin C能够在 36°C,则将被认为不适合于CytoTrap双杂交   系统。
        2. 只有诱饵质粒不导致宿主的生长 酵母细胞在36℃的共转化试验中可能适用 使用CytoTrap双杂交系统的交互伙伴的屏幕。

  2. 生成CytoTrap双杂交cDNA文库
    可以使用来自各种生物体的mRNA构建CytoTrap双杂交cDNA文库。这里我们使用来自模拟植物拟南芥的mRNA作为实例。简言之,从野生型拟南芥植物(Col-0)的叶组织中提取和纯化mRNA,然后使用含有寡核苷酸引物的杂交寡核苷酸(dT)接头引物合成第一链cDNA, em> Xho I限制性位点和高保真逆转录酶。在合成第一链cDNA期间使用5-甲基dCTP代替常规dCTP,并且所得cDNA中的甲基化Xho I位点将对Xho I消化是有弹性的。使用DNA聚合酶I合成第二链cDNA。含有粘性EcoR I末端和平端(参见注释1中的序列)的衔接头与抛光的双链cDNA连接,然后cDNA用Xho I消化。将所得的在5'端含有Eco RI并在3'端含有Xho I的cDNA连接到通过Eco RI预切割的pMyr载体中,因此cDNA以由Gal1启动子(P GAL1 )驱动的有意义的表达取向插入。 cDNA文库 携带在pMyr质粒中(质粒图: http://www.chem-agilent.com /pdf/strata/217438.pdf )然后转化为高效率的E。大肠杆菌 XL10-Gold Kan细胞用于繁殖和储存。详细过程如下所述。
    cDNA合成:
    1. mRNA纯化。来自4周龄拟南芥Col-0的叶样品(〜2g)  收获植物,从叶组织提取总RNA 使用RNeasy Plant Mini Kit。然后从1mg的纯化的聚(A)mRNA纯化  总RNA,使用用于cDNA合成的Oligotex mRNA Mini试剂盒(Zhang et al al。,2014)。
    2. 第一链cDNA合成使用高保真逆转录酶AccuScript进行
      1. 将以下试剂加入无RNase的微量离心管中:
        10×第一链缓冲液:5μl
        第一链甲基核苷酸混合物:3μl
        接头 - 引物(1.4μg/μl):2μl
        核糖核酸酶核糖核酸酶抑制剂(40 U /μl):1微升
        mRNA:等于5μg
        DEPC处理的水:将体积调至50μl
      2. 在室温下孵育反应10分钟以使引物与模板退火
      3. 加入3微升AccuScript逆转录酶的反应,孵育反应在42℃下2小时。
    3. 第二链cDNA合成
      1. 将第一链cDNA反应管置于冰上,并向管中加入以下试剂:
        10x第二链缓冲液:20μl
        第二链dNTP混合物:6μl
        无菌ddH 2 O:139μl
        核糖核酸酶H(5U /μl):1μl
        DNA聚合酶I(9.0U /μl):11μl
      2. 轻轻混合反应物并旋转反应。
      3. 在16℃孵育3小时,然后将管置于冰上。 的 得到的双链cDNA(ds cDNA)应该适合适配子 结扎。
    4. 使cDNA末端平齐并连接EcoR I接头
      1. 加入以下试剂至第二链cDNA合成,并在72℃孵育30分钟。
        10mM dNTP混合物:4μl
        DNA聚合酶(NEB):4μl
      2. 加入200μlUltraPure TM苯酚:氯仿:异戊醇(25:24:1) 到反应并涡旋混合。 在室温下将反应物在14,000×g下旋转5分钟,然后转移上层(〜200 μl)至新管。
      3. 加入200μl氯仿并涡旋。 向下旋转 在14,000×g下在室温下反应5分钟,然后 转移上层(〜200微升)到一个新的管。
      4. 加入以下试剂沉淀cDNA,涡旋并保持反应在-20℃过夜。
        3 M乙酸钠:20μl
        100%(v/v)乙醇:400μl/
      5. 在4℃下在14,000×g下旋转cDNA,持续60分钟。
      6. 小心除去上清液,用70% 乙醇(v/v)。 在室温下以14,000×g离心沉淀2分钟 温度。 取出乙醇,在室温下干燥沉淀。
      7. 将沉淀重悬于8μlEcoR I接头中,并在4℃下孵育1小时以确保cDNA的完全溶解。
      8. 将以下试剂加入具有cDNA和Eco RI衔接子的管中 使体积达到10μl,并在16℃下孵育反应物 过夜。
        10×T4连接酶缓冲液(NEB):1μl
        T4连接酶(NEB):1μl
      9. 孵育管在70℃下30分钟终止反应。
    5. 磷酸化Eco RI末端。
      1. 在室温下将反应冷却10分钟
      2. 将以下试剂加入试管中,并在37℃孵育1小时。
        10x反应缓冲液(NEB):1μl
        无菌ddH 2 O:8μl
        T4多核苷酸激酶(NEB):2μl
      3. 将管在70℃下孵育30分钟以终止反应
      4. 将反应在室温下冷却10分钟,然后加入 以下试剂加入到反应中,在37℃下孵育3小时 10x反应缓冲液(NEB):5μl
        无菌ddH 2 O:22μl
        Xho I(NEB):3μl
      5. 沉淀cDNA通过加入以下试剂到管中,并在-20℃孵育过夜 10x STE缓冲液:5μl
        100%(v/v)乙醇:125μl
      6. 在4℃下在14,000×g下旋转反应60分钟。 去除 上清并在室温下干燥沉淀。 溶解沉淀 在100μl无菌ddH 2 O中。
    6. 用CHROMA SPIN TM + TE-400柱纯化ds cDNA。 (如果没有指定,则在室温下进行。)
      1. 通过颠倒柱几次重悬浮凝胶矩阵。 去掉 顶盖并取下底部的断开端。 放置 柱在2-ml收集管中
      2. 在700×g离心5分钟,然后取出收集管和缓冲液。
      3. 将柱子放到一个新的收集管,并添加100微升的cDNA样品到凝胶矩阵的顶部表面的中心。
      4. 以700×g离心5分钟,收集含有cDNA(大小> 200bp)的洗脱溶液。
      5. 通过向洗脱的溶液中加入以下试剂沉淀cDNA,并在-20℃冷冻器中温育过夜。
        3 M乙酸钠(pH5.2):10微升
        冰冷的100%乙醇:250μl
      6. 在4℃下以14,000×g离心30分钟。 丢弃 上清液,加入360μl70%乙醇,反转数次, 然后在4℃下以14,000×g离心5分钟。 除去上清液 并在室温下干燥沉淀。 重悬在5微升的cDNA 无菌ddH 2 O。
    7. 将cDNA导入pMyr载体并转化入大肠杆菌。
      1. 通过加入以下试剂建立T4连接反应,在16℃温育过夜。
        10x连接酶缓冲液(NEB):2.5μl
        重悬cDNA(1μg):Xμl
        pMyr载体(通过 Eco RI/ Xho I,500ng):5μl
        T4 DNA连接酶(NEB):2.5μl
        ddH 2 O:将音量调到25μl
      2. 将所有连接反应转化至XL10-Gold Kan超级反应体系   细胞。 根据使用2μl连接反应每次转化 制造商的说明。
      3. 平板变换 包含氯霉素(25μg/ml)的150-mm LB平板,约50-100 板总数。 传播适当的变换量,这样 每个板可以具有30,000个菌落。 在37℃孵育过夜 允许菌落生长。 菌落总数应为3 百万。
      4. 每板加入5 ml LB培养基以刮除细菌   并使用另外的1ml LB培养基洗涤每个平板。 将所有的细菌在一个烧杯中混合,轻轻地混合。等分10 ml细菌培养在15-ml离心管中并储存在-80℃。 从每个10ml等分试样中纯化质粒用于CytoTrap双杂交筛选。

  3. 筛选CytoTrap双杂交cDNA文库
    将诱饵和cDNA文库质粒共转化到温度敏感宿主菌株cdc25H中。将转化反应物在室温下涂布在含有葡萄糖的SC/-LU平板上以使阳性转化体生长,然后将转化体复制到含有半乳糖的SC/-LU平板上,并在36℃温育以选择表达诱饵和潜在的相互作用配偶体基因。进一步分析阳性克隆以确认诱饵特异性蛋白质相互作用。图书馆筛选流程图如图1所示。
    1. 将诱饵和cDNA文库质粒共转化到宿主菌株cdc25H中 该步骤按照Yeastmaker TM supam Yeast Transformation建议进行  系统2手册除了所有的酵母菌培养步骤都必须 在室温下进行,除非另有规定降低速率 的cdc25H的回复突变。转化反应扩散开  SC/-LU板(150-mm),并保持在室温 持续3至4天,直到菌落生长至直径2-3mm。这是理想的 在每个板上有〜3,000个菌落,并且有必要在筛选 至少100万殖民地。
    2. 复制板生长的殖民地  含有半乳糖的SC/-LU板(150-mm),并将板留在 室温下1小时,然后移至选择温度  为36℃,保温3〜6天。含有推定的菌落 相互作用的合作伙伴和回复突变体将长大,它是 当它们直径为2-3mm时需要拾取菌落 在选择期间,并且不等待在选择结束时拾取
    3. 挑取在36℃下含有半乳糖的SC/-LU平板上长出的菌落  ℃,并将菌落溶解在25μl无菌dH 2 O中。点5微升的酵母 悬浮于四个板:含有葡萄糖和两个SC/-LU的两个SC/-LU 含有半乳糖。 孵育一个含有葡萄糖的SC/-LU板 一个含有半乳糖的SC/LU板在室温下,另一个 两个板在36℃。
      注意:
      1. 如果存在更多的酵母菌落,则培养板的数目应该放大。
      2. 如图1所示,仅在SC/-LU上生长的酵母菌落 而不是在36℃含有葡萄糖的SC/LU上 作为进一步分析的主要候选人。
      3. 在36℃下在半乳糖和葡萄糖平板上生长的酵母菌落 是温度回复突变体,并且它们在选择性的生长 温度是由cdc25H突变基因的回复突变引起的 野生型基因,因此它们在36℃的生长不是半乳糖依赖性的。
      4. 大部分的假阳性将是温度恢复   突变体并且可以通过测试半乳糖依赖性容易地鉴定 在36℃生长。


        图1. CytoTrap双杂交文库筛选 程序。 pSos-诱饵与a共转化到酵母菌株cdc25H中   在pMyr中的cDNA文库。 在SC/-LU上选择阳性转化体 (葡萄糖)平板。 然后复制菌落 在SC/-LU(半乳糖)平板上并在36℃温育3至6天。 在36℃生长的菌落进一步测试半乳糖和 诱饵特异性生长以鉴定真实的相互作用克隆。

  4. 分析积极互动候选人
    来自文库筛选的主要阳性候选物需要进一步测试,以查看相互作用是否是诱饵特异性的,并且需要鉴定猎物基因的性质以进行功能分析。
    1. 从阳性候选人隔离猎物质粒。
      1. 在2ml含有葡萄糖的液体SC/-LU中接种阳性酵母候选物,并在30℃振荡2天。
      2. 转移1.5毫升酵母培养物到1.7毫升离心管,并旋转   将细胞以14,000×g离心1分钟。 取出上清液并加入   200μl酵母裂解液,通过涡旋重悬细胞。
      3. 加 将200μlUltraPure TM苯酚:氯仿:异戊醇(25:24:1,v/v)和   0.3g酸洗玻璃珠,每次大力涡旋2分钟 样品。
      4. 在室温下以14,000×g离心5分钟, 并将顶层转移到新的微量离心管中。 加上 以下试剂加入每个管中,并将管在-20℃保持30分钟 min。
        3 M乙酸钠(pH 5.2):20μl
        冰冷的100%乙醇:500μl
      5. 在4℃下以14,000×g离心沉淀10分钟。 去除 上清液,并用70%乙醇洗涤沉淀。 旋转颗粒 在14,000×g下在室温下温育5分钟。 取出上清液 在室温下干燥沉淀。
      6. 在50中重悬沉淀 μl无菌ddH 2 O,然后使用1-2μl酵母质粒DNA进行转化 10-β电感受态大肠杆菌感受态细胞。 传播 在LB /氯霉素琼脂平板上进行转化反应。 置于37℃ 过夜。
      7. 挑取单个菌落并接种 LB /氯霉素液体培养基中37℃培养过夜。 净化 质粒用于进一步分析。
    2. 酵母共转化测试诱饵特异性蛋白质 - 蛋白质相互作用
      1. 用pSos诱饵或pSos空载体将纯化的候选猎物质粒转化到宿主菌株cdc25H中。
      2. 在室温下将转化体接种在含有葡萄糖的SC/-LU上 温度4天。 拿起三个独立的殖民地并转移 到含有葡萄糖的SC/-LU和含有SC/-U的半乳糖平板,   在36℃孵育3至6天。
      3. 只有猎物质粒显示   特异性地与pSos诱饵,不与pSos空载体相互作用 保持用于序列分析。
      4. 通过DNA测序确定cDNA插入物的身份。

笔记

  1. 含有内聚EcoR I末端和平端的衔接子序列:
    5'-OH-AATTCGGCACGAGG-3'
    3'-GCCGTGCTCCp-5'

食谱

  1. 酵母裂解溶液(100ml) 2ml Triton X-100 1克SDS
    0.58g NaCl
    1ml 1M Tris-HCl(pH8.0)
    200μl0.5M EDTA(pH 8.0)
    使用无菌dH 2 O 2将体积加至100ml 在室温下贮存
  2. YPDA/YPAD(1 L)
    10g酵母提取物
    20g Bacto 蛋白胨
    20g葡萄糖
    40mg腺嘌呤硫酸盐
    使用无菌dH 2 O使体积为1L,并调节至pH 5.7-5.8。
    如果准备板,加入15克琼脂
    在121℃高压灭菌20分钟
  3. SC/-LU(葡萄糖,1L) 6.7g无氨基酸的酵母氮源
    20g葡萄糖
    0.6 g DO补充-His/-Leu/-Trp/-Ura
    0.02克组氨酸 0.05 g色氨酸
    使用无菌dH 2 O使体积为1L,并调节至pH 5.7-5.8。
    如果准备板,加入15克琼脂
    在121℃高压灭菌20分钟
  4. SC/-LU(半乳糖,1L) 6.7g无氨基酸的酵母氮源
    20g半乳糖 10g棉子糖 0.6g DO补充剂-His/-Leu/-Trp/-Ura
    0.02克组氨酸 0.05 g色氨酸
    使用无菌dH 2 O使体积为1L,并调节至pH 5.7-5.8。
    如果准备板,加入15克琼脂
    在121℃高压灭菌20分钟
  5. 10×STE缓冲液(1L)
    58.44克NaCl
    200ml 1M Tris-HCl(pH7.5)
    200ml 0.5M EDTA
    使用无菌dH 2 O 2将体积加至1L

致谢

该研究由国家科学基金会授予B.W.的1021344号资助。 Poovaiah和中国国家科学基金会授予U1130304给Liqun Du。 我们感谢华盛顿州立大学的Lorie Mochel和Ade Snider帮助准备手稿。

参考文献

  1. Aronheim,A.,Zandi,E.,Hennemann,H.,Elledge,S.J.and Karin,M。(1997)。 通过检测蛋白质 - 蛋白质相互作用的新方法分离AP-1阻遏物。 Mol Cell Biol 17(6):3094-3102。
  2. Li,N.,Batzer,A.,Daly,R.,Yajnik,V.,Skolnik,E.,Chardin,P.,Bar-Sagi,D.,Margolis,B.and Schlessinger, 鸟嘌呤核苷酸释放因子hSos1结合Grb2并将受体酪氨酸激酶连接至Ras信号。 a> Nature 363(6424):85-88
  3. Zhang,L.,Du,L.,Shen,C.,Yang,Y.and Poovaiah,B.W。(2014)。 通过泛素介导的调节Ca(2+) - 钙调蛋白-ATSR1/CAMTA3调节植物免疫 植物J 78(2):269-281。
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How to cite this protocol: Zhang, L., Du, L. and Poovaiah, B. W. (2014). CytoTrap Two-Hybrid Screening Assay. Bio-protocol 4(23): e1310. DOI: 10.21769/BioProtoc.1310; Full Text



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(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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