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May 2018

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In vitro Protein-DNA Binding Assay (AlphaScreen® Technology)
蛋白质-DNA结合的体外检测法 (AlphaScreen® Technology)   

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Abstract

Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our in vitro transcription/translation system and AlphaScreen® technology. The in vitro transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen® system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions in vitro. Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.

Keywords: Transcription factor (转录因子), In vitro transcription/translation (体外转录翻译), FLAG-tag (FLAG-tag), Protein-DNA interaction (蛋白DNA相互作用), AlphaScreen (AlphaScreen), DNA binding (DNA结合)

Background

Upon exposure to abiotic and biotic stresses, major transcriptional changes are induced in plants to help them adapt to these environmental stimuli. Transcriptional regulation is important not only for stress responses but also for plant development. In general, one transcription factor regulates an array of target genes by recognizing its corresponding cis-regulatory elements. Thus, to study the physiological role of transcription factors, identification of their specific binding sequences is a major challenge. Recently, chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) has enabled researchers to determine the genome-wide locations of a transcription factor-DNA interaction. However, the precise binding capability should be further confirmed by in-depth biochemical experiments.

For the past several decades, electrophoretic mobility shift assay (EMSA) and transient reporter assay using viable cells have been widely used to determine protein-DNA interactions. For EMSA, a protein-DNA complex is subjected to electrophoresis followed by the autoradiographic or immunodetection of the band shift. For transient assay, protoplasts or tissues are transformed with a plasmid that consists of a promoter sequence harboring candidate cis-regulatory elements fused with a reporter gene encoding such as luciferase or green fluorescent protein (Chalfie et al., 1994). Although these are powerful methods to investigate the direct binding site of a transcription factor and its transcriptional activity, they are time-consuming and thus cannot be applicable to a comprehensive analysis.

On the other hand, Amplified Luminescence Proximity Homogenous Assay (Alpha) technology is an emerging alternative method to effectively detect protein-DNA interaction (AlphaScreen®). AlphaScreen assay uses two types of small beads (250 nm in diameter), Acceptor and Donor beads, which are specifically designed to associate with either a target protein fused with a protein tag or a biotinylated cis-regulatory element. When the protein-DNA interaction occurs, illumination at 680 nm releases singlet oxygen from donor beads, which subsequently transfers energy to acceptor beads to generate light at 520-620 nm. Since the half-life of singlet oxygen is only 4 µsec, the interaction is not detected without close proximity (see also web site: www.perkinelmer.com/alphascreen). Since AlphaScreen® shows high sensitivity and low background in a microplate format, it can adapt to a high-throughput screening platform.

Compared to EMSA and transient assay, AlphaScreen® is considerably easier to handle and quicker for the detection of protein-DNA interaction. Moreover, we have developed a highly efficient way to synthesize proteins in vitro (Nomoto and Tada, 2018a and 2018b). This method enables us to synthesize proteins that are difficult to obtain in cell-based production systems such as E. coli. Furthermore, this method is highly time efficient because the DNA template for in vitro transcription can be made by the second round of PCR using any type of cloning vectors. To take these advantages, we describe a protocol from protein synthesis to AlphaScreen® assay for detecting protein-DNA interactions. We confirm that our method contributes to the understanding of biological roles of transcription factors function in diverse signaling pathways.

Materials and Reagents

  1. Aluminum foil
  2. 1.5 ml microcentrifuge tubes
  3. 0.2 ml 8 strip PCR tube and cap strips
  4. 5’-biotinylated and non-biotinylated 50 base single-strand DNA and complementary unmodified 50 base single-strand DNA (Eurofins, Japan)
  5. Plasmid containing cDNA region of the gene of interest
  6. Gene-specific primers for 1st and 2nd PCR (see Table 1)
  7. Ultrapure water (Milli-Q water)
  8. KOD-Plus-Neo (including 10x PCR buffer, 2 mM dNTPs, 25 mM MgSO4 and KOD-Plus-Neo DNA polymerase) (TOYOBO, catalog number: KOD-401)
  9. Agarose (VWR, catalog number: 0710-500g) 
  10. Quick-Load Purple 1 kb Plus DNA ladder (0.1-10.0 kb) (New England BioLabs, catalog number N0550S)
  11. Ethidium bromide (Wako Pure Chemical, catalog number: 547-00101)
  12. RNase-free water
  13. 4 M ammonium acetate diluted with RNase-free water
  14. 99.5% ethanol (Wako Pure Chemical, special grade, catalog number: 057-00451)
  15. In Vitro Transcription/Translation Kit (NUProtein, catalog number: PSS3050)
  16. Polyacrylamide gel
  17. AlphaScreen FLAG® (M2) Detection kit (PerkinElmer, catalog number: 6760613C)
  18. AlphaPlate-384, 384-well plate (PerkinElmer, catalog number: 6005350)
  19. Tween® 20 (TCI, catalog number: T0543)
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058-5G)
  21. Polydeoxyadenylic acid-Polythymidylic acid (dAdT) (Sigma-Aldrich, catalog number: P9764-5UN)
  22. DL-dithiothreitol (DTT) (Wako Pure Chemical, catalog number: 047-08973)
  23. Tris (hydroxymethyl) aminomethane (Wako Pure Chemical, catalog number: 207-06275)
  24. Glacial acetic acid (Wako Pure Chemical, catalog number: 518-33985)
  25. Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865)
  26. Glycerol (Wako Pure Chemical, catalog number: 075-00616)
  27. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical, catalog number: 191-07145)
  28. Bromophenol blue (Wako Pure Chemical, catalog number: 021-02911)
  29. Xylene cyanolFF (Wako Pure Chemical, catalog number: 244-00461)
  30. 100x TAE buffer (see Recipes)
  31. 10x loading dye (see Recipes)

For SDS-PAGE and Western blotting

  1. Nitrocellulose membrane (GE Healthcare, catalog number: 10600007)
  2. WhatmanTM 3 MM Chr Chromatography Paper (GE Healthcare, catalog number: 05-714-5)
  3. Hybridization bag
  4. MXJB III Film (IBI, catalog number: 6567291)
  5. Precision Plus ProteinTM Dual color standards (Bio-Rad Laboratories, catalog number: 1610374)
  6. SDS polyacrylamide gel
  7. Methanol (Wako pure chemical, special grade, catalog number: 131-01826)
  8. Anti-DYKDDDDK tag antibody (diluted 1:2,000 in blocking buffer) (Wako Pure Chemical, catalog number: 014-22383)
  9. Goat anti-mouse IgG-HRP (diluted 1:1,000 in blocking buffer) (Cosmo Bio, catalog number: 1030-05)
  10. SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34577)
  11. Tris (hydroxymethyl) aminomethane (Wako Pure Chemical, catalog number: 207-06275)
  12. SDS (Wako Pure Chemical, catalog number: 191-07145)
  13. Bromophenol blue (Wako Pure Chemical, catalog number: 021-02911)
  14. Xylene cyano lFF (Wako Pure Chemical, catalog number: 244-00461)
  15. NaCl (Wako Pure Chemical, catalog number: 191-01665)
  16. Na2HPO4 (Wako Pure Chemical, catalog number: 197-02865)
  17. KCl (Wako Pure Chemical, catalog number: 163-03545)
  18. KH2PO4 (Wako Pure Chemical, catalog number: 169-04245)
  19. Skimmed milk powder (Wako Pure Chemical, catalog number: 190-12865)
  20. 10x running buffer (see Recipes)
  21. 4x SDS sample buffer (see Recipes)
  22. Transfer buffer (see Recipes)
  23. 10x PBS (see Recipes)
  24. Blocking buffer (see Recipes)

Equipment

  1. Pipettes
  2. PCR thermal cycler (Thermo Fisher Scientific, model: Veriti 200)
  3. Refrigerated centrifuge (Hitachi, model: himac CF 15R)
  4. Vortex mixer
  5. Block incubator (You can use a water incubator instead of block incubator.)
  6. Wonder Shaker (NISSIN, model: NA-4X)
  7. Multi Shaker (Tokyo Rikakikai Co., Ltd., model: MMS-120H)
  8. Heat Sealer (Taiyo Electric Co., Ltd., model: HS-400)
  9. Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, catalog number: 170-3940)
  10. Fuji Medical Film Processor (FUJIFILM Medical, model: FPM100)
  11. X-ray film cassette
  12. EnSpireTM Alpha Plate Reader (PerkinElmer, model: 2390-00000)

Note: Equipment #5 to #11 are shown for your reference for SDS-PAGE and Western blotting.

Procedure

  1. In vitro transcription/translation of a transcription factor using AlphaScreen®
    1. Preparation of DNA template for in vitro transcription/translation
      To attach a protein tag, in this case, “FLAG®-tag” to N-terminus of a target transcription factor, perform the two-step PCR method. All primers for making transcription templates are shown in Table 1.
      Note: For details, please refer to articles published in Genes Cells (Nomoto and Tada, 2018a) and Methods Mol Biol (Nomoto and Tada, 2018b) which include all the procedures for in vitro transcription/translation.

      Table 1. Primer sequences for transcription template PCR


      1. Prepare the following 1st PCR mix (total volume: 50 µl/sample) on ice:
        5 µl of 10x PCR buffer for KOD-Plus-Neo
        5 µl of 2 mM dNTPs
        3 µl of 25 mM MgSO4
        1 µl of KOD-Plus-Neo
        1 µl of plasmid (1 ng/µl)
        1 µl of 10 µM 1st-gene-specific-NF primer
        1 µl of 10 µM 1st-gene-specific-NR primer
        Add 33 µl of ultrapure water to make up the volume of the reaction to 50 µl
        Note: You can use any plasmid containing cDNA region of the gene of interest. In this case, cDNA region cloned into pENTR/D-TOPO was used as a 1st PCR template.
      2. Run the following 1st PCR
        Step 1: 94 °C for 5 min
        Step 2: 30 cycles of
        a) 98 °C for 10 s
        b) 55 °C for 30 s
        c) 68 °C for 3 min (30-60 s/kb)
        Step 3: 72 °C for 2 min
        Step 4: 4 °C until agarose gel electrophoresis
        Note: Temperature in this step (hybridization step) depends on primer sequences. You can change the temperature for this step according to normal PCR methods. It should be between 55 °C and 65 °C.
      3. Run 2 µl of the 1st PCR sample on agarose gel (1.5% [w/v] in 1x TAE buffer) electrophoresis to check the quantity and size of PCR amplicon (Figure 1).
        Note: If multiple PCR amplicons are detected in single PCR, gel extraction of a target band is recommended.
      4. Prepare the following 2nd PCR mix (total volume: 50 µl/sample) on ice:
        5 µl of 10x PCR buffer for KOD-Plus-Neo
        5 µl of 2 mM dNTPs
        3 µl of 25 mM MgSO4
        1 µl of KOD-Plus-Neo
        1 µl of 1st PCR sample
        1 µl of 10 µM 2nd-T7-NF2 primer
        1 µl of 100 nM 2nd-protein tag(FLAG)-NF1 primer
        1 µl of 10 µM 2nd-3’ SU-NCR2 primer
        1 µl of 100 nM 2nd-3’ SU-NR1 primer
        Add 31 µl of ultrapure water to make up the volume of the reaction to 50 µl
      5. Run the following 2nd PCR
        Step 1: 98 °C for 1 min
        Step 2: 10 cycles of
        a) 98 °C for 10 s
        b) 60 °C for 1 min
        c) 68 °C for 3 min (30-60 s/kb)
        Step 3: 30 cycles of
        a) 98 °C for 10 s
        b) 60 °C for 15 s
        c) 68 °C for 3 min (30-60 s/kb)
        Step 4: 72 °C for 2 min
        Step 5: 4 °C until agarose gel electrophoresis
      6. Run 2 µl of the 2nd PCR sample on agarose gel (1.5% [w/v] in 1x TAE buffer) electrophoresis to check the quantity and size of PCR amplicon (Figure 1).
        Note: If multiple PCR amplicons are detected in single PCR, gel extraction of a target band is recommended. One transcription reaction requires 25 µl of purified 2nd PCR amplicon containing 300 to 600 ng DNA.


        Figure 1. Agarose gel electrophoresis of products from the 1st PCR, 2nd PCR, and transcription products. Lane 1: DNA marker (2-log ladder), lane 2: 1st PCR product, lane 3: 2nd PCR product, lane 4: transcription reaction product. MYB30 coding region cloned into pENTR/D-TOPO vector is used as the template for 1st PCR. MYB30 coding region is 969 base pairs.

    2. In vitro transcription
      Perform In vitro transcription/translation reaction using NUProtein kit following the manufacturer’s protocol.
      1. Prepare the following transcriptional master mix (for one sample) on ice:
        2.5 µl of 10x Transcription Buffer (provided in the kit)
        1.25 µl of 0.1 M DTT (provided in the kit)
        2.5 µl of 25 mM NTPs (provided in the kit)
        1 µl of T7 RNA polymerase (provided in the kit)
        Add 15.25 µl of RNase-free water
        Note: If RNA degradation occurs, an RNase inhibitor should be added to the transcription mix.
      2. Dispense 22.5 µl of the above transcription mix to each new tube (RNase-free tube recommended) and add 2.5 µl of 2nd PCR product carefully (Purification of 2nd PCR products is not necessarily required).
        Note: NUProtein kit contains positive control DNA (2nd PCR amplicon) which can be used as a positive control for the in vitro transcription reaction.
      3. Incubate the sample at 37 °C for 3 h in a block incubator.
        Note: Incubation time depends on the type and length of mRNA, and may vary from 30 min to 3 h. Water incubator can also be used instead of block incubator.
      4. Run 1 µl of the transcription reaction sample can be loaded on agarose gel (1.5% [w/v] in 1x TAE buffer) electrophoresis to check the quantity and quality of mRNA (Figure 1). 
      5. Add 10 µl of 4 M ammonium acetate and 100 µl of 99.5 % ethanol per sample (total volume: 124 µl/sample.
      6. Mix the sample by gentle vortexing or tapping and incubate at -20 °C for 20 min after centrifugation for a few seconds.
      7. Centrifuge the sample at 17,700 x g for 20 min at 4 °C and discard the supernatant carefully.
      8. Repeat centrifugation for few seconds to remove the residual solution completely using a pipette.
      9. Dry the pellet by keeping the tube lid open for about 10 min.
      10. Add 70 µl of RNase-free water per sample and incubate at RT for about 15 min.
      11. Dissolve the pellet well by gentle tapping and centrifuging for a few seconds.
    3. In vitro protein synthesis
      1. Prepare the following translation reaction master mix (for one sample) into 1.5 ml tube on ice:
        20 µl of wheat germ extract (WGE) (provided in the kit)
        20 µl of amino acid mixture (AAM) (provided in the kit)
      2. Mix the translation reaction master mix gently, and incubate at RT for 15 min.
        Note: Preincubation of WGE and AAM may enhance the translation efficiency.
      3. Add 40 µl of the translation reaction mix to 70 µl of prepared mRNA carefully (total volume: 110 µl/sample).
        Note: Do not make bubbles during additions. Make AlphaScreen® negative control by adding 40 µl of the translation reaction mix to 70 µl of RNase-free water.
      4. Incubate the sample at 16 °C for 10 h without agitation.
      5. Centrifuge the sample at 17,700 x g for 10 min at 4 °C and transfer the supernatant (approximately 118 µl) to a new tube.
      6. Mix 1 µl of the in vitro-synthesized protein, 4 µl of 4x SDS sample buffer, 1 µl of 2 M DTT, and 6 µl of water in a new tube.
        Note: Remaining protein sample can be stored at -80 °C. We recommend flash-freezing the sample using liquid N2. Repeated freeze-thaw cycles decrease the activity of synthesized proteins. When using frozen protein for any assay, quickly thaw the sample by hand.
      7. Heat protein sample at 70 °C for 20 min and perform SDS-PAGE in a 10% (w/v) polyacrylamide gel and confirm the synthesized target protein by western blotting with anti-DYKDDDDK tag antibody.

  2. AlphaScreen® assay
    1. Preparation of double-stranded DNA probes by annealing
      For AlphaScreen®, detecting the interaction between protein and DNA, prepare biotinylated (target sample) and non-biotinylated (control for the detection) 50 bp DNA probes. For obtaining stable AlphaScreen® results, we recommend designing the DNA probes that contain putative DNA binding sequence of the target transcription factor in the central region (11 bp to 40 bp from 5’ end of the probe [Figure 2A]).
      1. Mix 20 µl of 20 µM biotinylated 50 bp single-strand DNA (or 20 µl of 20 µM non-biotinylated 50 bp single strand DNA) and 20 µl of 20 µM complementary unmodified 50 bp single-strand DNA in 1.5 ml tube (total volume: 40 µl).
      2. Incubate the DNA mixture at 95 °C for 10 min, let it cool down until it reaches RT.
        Note: Double-strand DNA probes can be stored at -20 °C.
      3. Check annealed double-stranded DNA probes by polyacrylamide gel (12% w/v) electrophoresis (Figure 2B).
        Note: At the same time prepare DNA probe for the negative control, which does not contain any putative DNA binding sequence of the target transcription factor. If required, also prepare the DNA probes that contain mutation in the target sequence.


      Figure 2. Schematic diagram of DNA probe for AlphaScreen ®. A. Schematic diagram of annealed double-strand DNA probe. Black lines and box indicate scaffold region (10 bases in length). Red lines and box indicate the region that contains putative DNA binding sequence of the target transcription factor. B. Polyacrylamide gel (10%) electrophoresis of DNA probes. Lane 1: DNA marker (2-log ladder), lane 2: 5’-biotinylated 50-base single-strand DNA, lane 3: Complementary 50-base single-strand DNA, lane 4: Annealed double-strand DNA. The polyacrylamide gel was stained with ethidium bromide after electrophoresis.

    2. AlphaScreen® assay with FLAG (M2) Detection kit
      1. Prepare the following protein-DNA binding mix (total volume: 13 µl/sample):
        2.5 µl of 10x control buffer [provided in FLAG (M2) Detection kit]
        2.5 µl of 0.1% (w/v) Tween® 20
        2.5 µl of 1% (w/v) BSA
        1 µl of 1 ng/µl dAdT
        Add 4.5 µl of ultrapure water.
        Note: Depending on the target transcription factor, dGdC should be used instead of dAdT.
      2. Dispense 13.0 µl of the protein-DNA binding mix to the 384-well plate and add 2 µl of 625 nM of double-stranded DNA probe and 4-fold diluted FLAG-tagged target transcription factor protein.
        Note: Also add 2 µl of 4-fold diluted negative control protein.
      3. Incubate protein-DNA binding mix at RT for 1 h.
        Note: Dilution range of synthesized protein depends on the feature of target transcription factor. Two to ten times dilution can be used for this assay. Incubation under dark condition is not necessary for this step.
      4. Add 4 µl of 40-fold diluted (with ultrapure water) acceptor beads and incubate at RT for 1 h.
        Note: Incubation under dark condition is not necessary for this step.
      5. Add 4 µl of 40-fold diluted (with ultrapure water) donor beads and incubate at RT for 1 h to 12 h in the dark (total volume: 25 µl).
        Note: To ensure that the plate is in the dark, wrap it with aluminum foil and remove it before keeping it in the Alpha plate reader.
      6. Detect signal by EnSpireTM Alpha Plate Reader. After the excitation at 680 nm, the emission wavelengths between 520 and 620 nm are measured as AlphaScreen® unit.

Data analysis

  1. Divide the signal intensity from the mix of biotinylated DNA probe by the signal intensity from the mix of non-biotinylated DNA probe. This signal can be used as “Relative signal intensity” for plotting a graph.
    Note: The signal intensity from negative controls (without target protein) usually shows very low signal. 
  2. Data from 3 technical replicates are combined for the statistical analysis, and the same experiment using different batches of proteins is repeated at least 3 times with similar results.
    Note: One example of AlphaScreen® result can be found in Figures 4B and 4C in Mabuchi et al., 2018. As an alternative way of representing data, you may also be able to show all the signals, including one from negative controls.

Notes

  1. In this protocol, we used protein that is synthesized by our in vitro transcription/translation system. However, one may use protein that is synthesized by other lab protocols (e.g., using bacteria, yeast, and any other systems).
  2. In this protocol, we use FLAG®-tagged protein. However, one may use other protein tags, (e.g., GST, 6-Histidine, c-myc, and HA) as long as detection kits for “AlphaScreen® Fusion Tag” are available from PerkinElmer (see also web site: www.perkinelmer.com/alphascreen).

Recipes

  1. 100x TAE buffer
    400 mM Tris-HCl
    400 mM glacial acetic aid
    10 mM EDTA, pH 8.0
  2. 10x loading dye
    50% (w/v) glycerol
    0.9% (w/v) SDS
    10 mM EDTA, pH 8.0
    0.05% (w/v) bromophenol blue
    0.05% (w/v) xylene cyanol
  3. 4x SDS sample buffer
    200 mM Tris-HCl, pH 6.8
    40% (w/v) glycerol
    8% (w/v) SDS
    0.08% (w/v) bromophenol blue
  4. 10x running buffer
    250 mM Tris
    1.92 M glycine
    1% (w/v) SDS
  5. Transfer buffer
    192 mM glycine
    20% (w/v) methanol
    25 mM Tris
  6. 10x PBS
    1.37 M NaCl
    81 mM Na2HPO4
    26.8 mM KCl
    14.7 mM KH2PO4
  7. Blocking buffer
    1x PBS
    1% (w/v) non-fat skimmed milk
    0.1% (w/v) Tween® 20

Acknowledgments

This work was supported by Ministry of Education, Culture, Sports, Science, and Technology Grant-in-Aid for Scientific Research on Innovative Areas 26113508 to H.T., JSPS KAKENHI (No. 13J10800) and Program for Leading Graduate Schools “Integrative Graduate Education and Research in Green Natural Sciences”, MEXT, Japan to M.N., Grant-in-Aid for Scientific Research on Innovative Areas (No. 23120520 and 25120718) from the Ministry of Education, Culture, Sports, Science and Technology (Japan), JSPS KAKENHI (No. 15H05956) to Y.T. In vitro protein synthesis was adapted from Nomoto and Tada, 2018a and 2018b and Mabuchi et al., 2018. AlphaScreen for protein-DNA interaction was adapted from Mabuchi et al., 2018.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Chalfie. M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263(5148): 802-805.
  2. Mabuchi, K., Maki, H., Itaya, T., Suzuki, T., Nomoto, M., Sakaoka, S., Morikami, A., Higashiyama, T., Tada, Y., Busch, W. and Tsukagoshi, H. (2018). MYB30 links ROS signaling, root cell elongation, and plant immune responses. Proc Natl Acad Sci U S A 115(20): E4710-E4719.
  3. Nomoto, M. and Tada, Y. (2018a). Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR. Genes Cells 23(1): 46-53.
  4. Nomoto, M. and Tada, Y. (2018b). Cell-free protein synthesis of plant transcription factors. Methods MolBiol 1830: 337-349.

简介

鉴定转录因子的特定DNA结合位点对于理解它们的功能是重要的。最近的技术允许我们通过染色质免疫沉淀结合高通量测序来研究全基因组体内结合位置。然而,为了进一步探索转录因子的结合基序,需要进行深入的生化分析。在这里,我们描述了基于我们的体外转录/翻译系统和AlphaScreen ®技术的组合的蛋白质-DNA相互作用的有效方案。 体外转录/翻译系统通过减轻繁琐的克隆步骤,支持有效且快速的蛋白质合成方式。此外,AlphaScreen ®系统提供了一个高度灵敏,快速,易于操作的平台,可用于研究体外蛋白质-DNA相互作用。因此,我们的方法在很大程度上有助于全面分析转录因子的生化特性。
【背景】在暴露于非生物和生物胁迫时,在植物中诱导主要的转录变化以帮助它们适应这些环境刺激。转录调控不仅对应激反应很重要,对植物发育也很重要。通常,一种转录因子通过识别其相应的顺式调节元件来调节靶基因阵列。因此,为了研究转录因子的生理作用,鉴定它们的特异性结合序列是一项重大挑战。最近,染色质免疫沉淀结合高通量测序(ChIP-seq)使研究人员能够确定转录因子-DNA相互作用的全基因组位置。然而,应通过深入的生化实验进一步证实精确的结合能力。

在过去的几十年中,电泳迁移率变动分析(EMSA)和使用活细胞的瞬时报告分析已被广泛用于确定蛋白质-DNA相互作用。对于EMSA,对蛋白质-DNA复合物进行电泳,然后进行放射自显影或免疫检测。对于瞬时测定,原生质体或组织用质粒转化,该质粒由含有与编码荧光素酶或绿色荧光蛋白的报告基因融合的候选顺式调节元件的启动子序列组成(Chalfie 等。, 1994年)。虽然这些是研究转录因子的直接结合位点及其转录活性的有效方法,但它们是耗时的,因此不适用于综合分析。

另一方面,放大发光邻近同源测定(Alpha)技术是一种有效检测蛋白质-DNA相互作用的新兴替代方法(AlphaScreen ®)。 AlphaScreen测定使用两种类型的小珠子(直径250nm),受体和供体珠子,其特别设计用于与蛋白质标签或生物素化的顺式调节元件融合的靶蛋白质。当蛋白质-DNA相互作用发生时,680nm处的光照从供体珠粒释放单线态氧,随后将能量转移到受体珠粒以产生520-620nm的光。由于单线态氧的半衰期仅为4微秒,因此如果没有紧密接近,则无法检测到相互作用(另请参见网站: www.perkinelmer.com/alphascreen )。由于AlphaScreen ®在微孔板格式中显示出高灵敏度和低背景,因此它可以适应高通量筛选平台。

与EMSA和瞬时测定相比,AlphaScreen ®更容易处理并且更快地检测蛋白质-DNA相互作用。此外,我们已经开发出一种高效合成蛋白质体外的方法(Nomoto和Tada,2018a和2018b)。该方法使我们能够合成在基于细胞的生产系统(例如 E)中难以获得的蛋白质。大肠杆菌。此外,该方法是高度时间有效的,因为用于体外转录的DNA模板可以通过使用任何类型的克隆载体的第二轮PCR进行。为了获得这些优势,我们描述了从蛋白质合成到AlphaScreen ®测定的用于检测蛋白质-DNA相互作用的方案。我们确认我们的方法有助于理解转录因子功能在不同信号通路中的生物学作用。

关键字:转录因子, 体外转录翻译, FLAG-tag, 蛋白DNA相互作用, AlphaScreen, DNA结合

材料和试剂

  1. 铝箔
  2. 1.5毫升微量离心管
  3. 0.2毫升8条PCR管和盖条
  4. 5'-生物素化和非生物素化的50碱基单链DNA和互补的未修饰的50碱基单链DNA(Eurofins,Japan)
  5. 含有目的基因的cDNA区域的质粒
  6. 用于第1和第2次PCR的基因特异性引物(见表1)
  7. 超纯水(Milli-Q水)
  8. KOD-Plus-Neo(包括10x PCR缓冲液,2 mM dNTPs,25 mM MgSO 4 和KOD-Plus-Neo DNA聚合酶)(TOYOBO,目录号:KOD-401)
  9. 琼脂糖(VWR,目录号:0710-500g) 
  10. 快速加载紫色1 kb Plus DNA梯(0.1-10.0 kb)(New England BioLabs,目录号N0550S)
  11. 溴化乙锭(Wako Pure Chemical,目录号:547-00101)
  12. 不含RNase的水
  13. 用不含RNase的水稀释4M乙酸铵
  14. 99.5%乙醇(Wako Pure Chemical,特殊等级,目录号057-00451)
  15. 体外转录/翻译试剂盒(NUProtein,目录号:PSS3050)
  16. 聚丙烯酰胺凝胶
  17. AlphaScreen FLAG ®(M2)检测试剂盒(PerkinElmer,目录号:6760613C)
  18. AlphaPlate-384,384孔板(PerkinElmer,目录号:6005350)
  19. Tween ® 20(TCI,目录号:T0543)
  20. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A2058-5G)
  21. 聚脱氧腺苷酸 - 多聚乙烯基酸(dAdT)(Sigma-Aldrich,目录号:P9764-5UN)
  22. DL-二硫苏糖醇(DTT)(Wako Pure Chemical,目录号:047-08973)
  23. 三(羟甲基)氨基甲烷(Wako Pure Chemical,目录号:207-06275)
  24. 冰醋酸(Wako Pure Chemical,目录号:518-33985)
  25. 乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
  26. 甘油(Wako Pure Chemical,目录号:075-00616)
  27. 十二烷基硫酸钠(SDS)(Wako Pure Chemical,目录号:191-07145)
  28. Bromophenol blue(Wako Pure Chemical,目录号:021-02911)
  29. 二甲苯cyanolFF(Wako Pure Chemical,目录号:244-00461)
  30. 100x TAE缓冲液(见食谱)
  31. 10倍装载染料(见食谱)

用于SDS-PAGE和Western印迹

  1. 硝酸纤维素膜(GE Healthcare,目录号:10600007)
  2. Whatman TM 3 MM Chr色谱纸(GE Healthcare,目录号:05-714-5)
  3. 杂交袋
  4. MXJB III Film(IBI,目录号:6567291)
  5. Precision Plus Protein TM 双色标准品(Bio-Rad Laboratories,目录号:1610374)
  6. SDS聚丙烯酰胺凝胶
  7. 甲醇(Wako纯化学品,特殊等级,目录号:131-01826)
  8. 抗DYKDDDDK标签抗体(在封闭缓冲液中1:2,000稀释)(Wako Pure Chemical,目录号:014-22383)
  9. 山羊抗小鼠IgG-HRP(在封闭缓冲液中1:1,000稀释)(Cosmo Bio,目录号:1030-05)
  10. SuperSignal TM West Pico PLUS化学发光底物(Thermo Fisher Scientific,目录号:34577)
  11. 三(羟甲基)氨基甲烷(Wako Pure Chemical,目录号:207-06275)
  12. SDS(Wako Pure Chemical,目录号:191-07145)
  13. Bromophenol blue(Wako Pure Chemical,目录号:021-02911)
  14. 二甲苯氰基lFF(Wako Pure Chemical,目录号:244-00461)
  15. NaCl(Wako Pure Chemical,目录号:191-01665)
  16. Na 2 HPO 4 (Wako Pure Chemical,目录号:197-02865)
  17. KCl(Wako Pure Chemical,目录号:163-03545)
  18. KH 2 PO 4 (Wako Pure Chemical,目录号:169-04245)
  19. 脱脂奶粉(Wako Pure Chemical,目录号:190-12865)
  20. 10x运行缓冲区(参见食谱)
  21. 4x SDS样品缓冲液(参见食谱)
  22. 转移缓冲区(见食谱)
  23. 10x PBS(见食谱)
  24. 阻塞缓冲区(见食谱)

设备

  1. 移液器
  2. PCR热循环仪(Thermo Fisher Scientific,型号:Veriti 200)
  3. 冷冻离心机(日立,型号:himac CF 15R)
  4. 涡旋混合器
  5. 块孵化器(您可以使用水培养箱代替块孵化器。)
  6. Wonder Shaker(NISSIN,型号:NA-4X)
  7. Multi Shaker(东京Rikakikai有限公司,型号:MMS-120H)
  8. 热封机(Taiyo Electric Co.,Ltd.,型号:HS-400)
  9. Trans-Blot ® SD半干电泳转移细胞(Bio-Rad Laboratories,目录号:170-3940)
  10. 富士医疗胶片处理器(FUJIFILM Medical,型号:FPM100)
  11. X光胶片盒
  12. EnSpire TM Alpha Plate Reader(PerkinElmer,型号:2390-00000)

注意:显示设备#5至#11供您参考SDS-PAGE和Western印迹。

程序

  1. 使用AlphaScreen ® 转录因子的体外转录/翻译
    1. 制备体外转录/翻译的DNA模板
      在这种情况下,将“FLAG ® -tag”连接到靶转录因子的N末端,进行两步PCR方法。用于制备转录模板的所有引物如表1所示。
      注意:有关详细信息,请参阅Genes Cells(Nomoto和Tada,2018a)和Methods Mol Biol(Nomoto和Tada,2018b)中发表的文章,其中包括体外转录/翻译的所有程序。

      表1.转录模板PCR的引物序列


      1. 在冰上准备以下第一次PCR混合物(总体积:50μl/样品):
        用于KOD-Plus-Neo的5μl10xPCR缓冲液 5μl2mM dNTP
        3μl25mMMgSO 4
        1μlKOD-Plus-Neo
        1μl质粒(1 ng /μl)
        1μl10μM第一基因特异性NF引物
        1μl10μM第一基因特异性NR引物
        加入33μl超纯水,使反应体积达到50μl
        注意:您可以使用任何含有目的基因cDNA区域的质粒。在这种情况下,克隆到pENTR / D-TOPO中的cDNA区域用作第一PCR模板。
      2. 运行以下第一次PCR
        步骤1:94℃5分钟
        第2步:30个循环的
        a)98°C持续10秒
        b)55°C 30秒
        c)68℃3分钟(30-60秒/ kb)
        步骤3:72°C保持2分钟
        步骤4:4℃直至琼脂糖凝胶电泳 注意:此步骤中的温度(杂交步骤)取决于引物序列。您可以根据常规PCR方法更改此步骤的温度。它应该在55°C和65°C之间。
      3. 在琼脂糖凝胶(1%TAE缓冲液中1.5%[w / v])电泳上运行2μl第一份PCR样品,检查PCR扩增子的数量和大小(图1)。
        注意:如果在单个PCR中检测到多个PCR扩增子,建议使用凝胶提取目标条带。
      4. 在冰上准备以下第二种PCR混合物(总体积:50μl/样品):
        用于KOD-Plus-Neo的5μl10xPCR缓冲液 5μl2mM dNTP
        3μl25mMMgSO 4
        1μlKOD-Plus-Neo
        1μl第一次PCR样品
        1μl10μM第二-T7-NF2引物
        1μl100nM第二蛋白标签(FLAG)-NF1引物
        1μl10μM第二-3'SU-NCR2引物
        1μl100nM 2nd-3'SU-NR1引物
        加入31μl超纯水,使反应体积达到50μl
      5. 运行以下第二次PCR
        步骤1:98°C 1分钟
        第2步:10个循环的
        a)98°C持续10秒
        b)60°C 1分钟
        c)68℃3分钟(30-60秒/ kb)
        第3步:30个循环的
        a)98°C持续10秒
        b)60°C,持续15秒
        c)68℃3分钟(30-60秒/ kb)
        步骤4:72℃2分钟
        步骤5:4℃直至琼脂糖凝胶电泳
      6. 在琼脂糖凝胶(1.5%[w / v]在1x TAE缓冲液中)电泳上运行2μl第二PCR样品,检查PCR扩增子的数量和大小(图1)。
        注意:如果在单次PCR中检测到多个PCR扩增子,建议使用凝胶提取目标条带。一个转录反应需要25μl纯化的第二PCR扩增子,含有300至600 ng DNA。


        图1.来自第1次PCR,第2次PCR和转录产物的产物的琼脂糖凝胶电泳。泳道1:DNA标记(2-log ladder),泳道2:第1次PCR产物,泳道3:第2次PCR产物,泳道4:转录反应产物。克隆到pENTR / D-TOPO载体中的 MYB30 编码区用作第一次PCR的模板。 MYB30 编码区是969个碱基对。

    2. 体外转录
      按照制造商的方案,使用NUProtein试剂盒进行体外转录/翻译反应。
      1. 在冰上准备以下转录主混合物(一个样品):
        2.5μl10x转录缓冲液(试剂盒中提供)
        1.25μl的0.1 M DTT(试剂盒中提供)
        2.5μl25mMNTP(试剂盒中提供)
        1μlT7RNA聚合酶(试剂盒中提供)
        加入15.25μl不含RNase的水
        注意:如果发生RNA降解,应在转录组合中加入RNase抑制剂。
      2. 将22.5μl上述转录混合物分配到每个新管(建议使用无RNase管)并仔细添加2.5μl第二PCR产物(不一定需要纯化第二PCR产物)。
        注意:NUProtein试剂盒含有阳性对照DNA(第2次PCR扩增子),可用作 体外 转录反应的阳性对照。
      3. 将样品在37℃下在块状培养箱中孵育3小时。
        注意:孵育时间取决于mRNA的类型和长度,可能在30分钟到3小时之间变化。也可以使用水培养箱代替块培养箱。
      4. 运行1μl转录反应样品可加载到琼脂糖凝胶(1.5%[w / v]在1x TAE缓冲液中)电泳,以检查mRNA的数量和质量(图1)。 
      5. 每个样品加入10μl4M乙酸铵和100μl99.5%乙醇(总体积:124μl/样品。
      6. 通过温和涡旋或轻拍混合样品,并在离心几秒后在-20℃下孵育20分钟。
      7. 将样品在17,700 x g 下于4°C离心20分钟,并小心弃去上清液。
      8. 重复离心几秒钟,用移液管完全除去残留的溶液。
      9. 通过保持管盖打开约10分钟来干燥颗粒。
      10. 每个样品加入70μl不含RNase的水,并在室温下孵育约15分钟。
      11. 通过轻轻敲打并离心几秒钟将颗粒充分溶解。
    3. 体外蛋白质合成
      1. 准备以下翻译反应主混合物(对于一个样品)在冰上的1.5ml管中:
        20μl小麦胚芽提取物(WGE)(试剂盒中提供)
        20μl氨基酸混合物(AAM)(试剂盒中提供)
      2. 轻轻混合翻译反应主混合物,在室温下孵育15分钟。
        注意:WGE和AAM的预孵育可以提高翻译效率。
      3. 将40μl翻译反应混合物小心地加入70μl制备的mRNA中(总体积:110μl/样品)。
        注意:添加时不要泡泡。通过向70μl不含RNase的水中添加40μl翻译反应混合物,使AlphaScreen ® 阴性对照。
      4. 将样品在16°C孵育10小时,无需搅拌。
      5. 将样品在17,700 x g 下在4℃下离心10分钟,并将上清液(约118μl)转移到新管中。
      6. 在新管中混合1μl体外 - 合成蛋白,4μl4xSDS样品缓冲液,1μl2M DTT和6μl水。
        注意:剩余的蛋白质样品可以在-80°C下储存。我们建议使用液体N 2 对样品进行快速冷冻。重复的冻融循环降低了合成蛋白质的活性。当使用冷冻蛋白进行任何测定时,请用手快速解冻样品。
      7. 将蛋白质样品在70℃加热20分钟,并在10%(w / v)聚丙烯酰胺凝胶中进行SDS-PAGE,并通过用抗DYKDDDD标签抗体进行蛋白质印迹确认合成的靶蛋白。

  2. AlphaScreen ®检测
    1. 通过退火制备双链DNA探针 对于AlphaScreen ®,检测蛋白质与DNA之间的相互作用,制备生物素化(目标样品)和非生物素化(检测对照)50 bp DNA探针。为了获得稳定的AlphaScreen ®结果,我们建议在中心区域设计含有目标转录因子的推定DNA结合序列的DNA探针(从探针的5'末端起11 bp到40 bp [图图2A])。
      1. 将20μl20μM生物素化的50 bp单链DNA(或20μl20μM非生物素化的50 bp单链DNA)和20μl20μM互补未修饰的50 bp单链DNA混合在1.5 ml管中(总体积) :40μl)。
      2. 将DNA混合物在95°C孵育10分钟,让其冷却至RT。
        注意:双链DNA探针可以在-20°C下保存。
      3. 通过聚丙烯酰胺凝胶(12%w / v)电泳检查退火的双链DNA探针(图2B)。
        注意:同时为阴性对照制备DNA探针,其不含任何推定的靶转录因子的DNA结合序列。如果需要,还要准备含有靶序列突变的DNA探针。


      图2. AlphaScreen ®的DNA探针示意图。 A.退火双链DNA探针的示意图。黑色线条和框表示支架区域(长度为10个碱基)。红线和框表示含有靶转录因子的推定DNA结合序列的区域。 B.DNA探针的聚丙烯酰胺凝胶(10%)电泳。泳道1:DNA标记(2-log梯),泳道2:5'-生物素化的50碱基单链DNA,泳道3:互补的50碱基单链DNA,泳道4:退火的双链DNA。电泳后,聚丙烯酰胺凝胶用溴化乙锭染色。

    2. 使用FLAG(M2)检测试剂盒进行AlphaScreen ®检测
      1. 准备以下蛋白质-DNA结合混合物(总体积:13μl/样品):
        2.5μl10x对照缓冲液[在FLAG(M2)检测试剂盒中提供]
        2.5μl0.1%(w / v)吐温® 20
        2.5μl的1%(w / v)BSA
        1μl1ng /μldAdT
        加入4.5μl超纯水。
        注意:根据目标转录因子,应使用dGdC代替dAdT。
      2. 将13.0μl蛋白质-DNA结合混合物分配到384孔板中,加入2μl625nM的双链DNA探针和4倍稀释的FLAG标记的靶转录因子蛋白。
        注意:另外加入2μl4倍稀释的阴性对照蛋白。
      3. 在室温下孵育蛋白质-DNA结合混合物1小时。
        注意:合成蛋白的稀释范围取决于靶转录因子的特征。稀释2至10倍可用于该测定。此步骤不需要在黑暗条件下孵育。
      4. 加入4μl40倍稀释(用超纯水)受体珠子,在室温下孵育1小时。
        注意:此步骤不需要在黑暗条件下孵育。
      5. 加入4μl40倍稀释(用超纯水)供体珠粒,在室温下在黑暗中孵育1小时至12小时(总体积:25μl)。
        注意:为确保印版在黑暗中,请用铝箔包裹并将其取下,然后再将其放入Alpha读板器中。
      6. 通过EnSpire TM Alpha Plate Reader检测信号。在680nm激发后,520和620nm之间的发射波长被测量为AlphaScreen ®单位。

数据分析

  1. 将来自生物素化DNA探针混合物的信号强度除以来自非生物素化DNA探针混合物的信号强度。该信号可以用作绘制图形的“相对信号强度”。
    注意:来自阴性对照(不含靶蛋白)的信号强度通常显示非常低的信号。 
  2. 将来自3个技术重复的数据组合用于统计分析,并且使用不同批次的蛋白质的相同实验重复至少3次,具有类似的结果。
    注意:AlphaScreen ®结果的一个例子可以在2018年Mabuchi 等人的图4B和4C中找到。,2018。作为表示数据的另一种方式,你也可能是能够显示所有信号,包括来自阴性对照的信号。

笔记

  1. 在该方案中,我们使用由我们的体外转录/翻译系统合成的蛋白质。然而,可以使用通过其他实验室方案合成的蛋白质(例如,使用细菌,酵母和任何其他系统)。
  2. 在该方案中,我们使用FLAG ®标记的蛋白质。但是,只要检测试剂盒“AlphaScreen ® Fusion标签,就可以使用其他蛋白质标签(例如,GST,6-Histidine,c-myc和HA) “可从PerkinElmer获得(另请参见网站: www.perkinelmer.com/alphascreen )。

食谱

  1. 100x TAE缓冲区
    400 mM Tris-HCl
    400 mM冰醋酸助剂
    10mM EDTA,pH 8.0
  2. 10倍装染料
    50%(w / v)甘油
    0.9%(w / v)SDS
    10 mM EDTA,pH 8.0
    0.05%(w / v)溴酚蓝
    0.05%(w / v)二甲苯蓝
  3. 4x SDS样品缓冲液
    200mM Tris-HCl,pH 6.8
    40%(w / v)甘油
    8%(w / v)SDS
    0.08%(w / v)溴酚蓝
  4. 10x运行缓冲区
    250 mM Tris
    1.92 M甘氨酸
    1%(w / v)SDS
  5. 转移缓冲区
    192 mM甘氨酸
    20%(w / v)甲醇
    25mM Tris
  6. 10x PBS
    1.37 M NaCl
    81 mM Na 2 HPO 4
    26.8 mM KCl
    14.7mM KH 2 PO 4
  7. 阻塞缓冲区
    1x PBS
    1%(w / v)无脂脱脂奶粉
    0.1%(w / v)Tween ® 20

致谢

这项工作得到了教育,文化,体育,科学和技术资助科学研究创新领域的支持26113508到HT,JSPS KAKENHI(第13J10800号)和领先研究生院计划“综合研究生教育和绿色自然科学研究“,MEXT,日本到MN,教育,文化,体育,科学和技术部(日本)的创新领域科学研究资助(编号23120520和25120718),JSPS KAKENHI (第15H05956号)至YT 体外蛋白质合成改编自Nomoto和Tada,2018a和2018b以及Mabuchi et al。,2018。用于蛋白质-DNA相互作用的AlphaScreen改编自Mabuchi et al。,2018。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Chalfie。 M.,Tu,Y.,Euskirchen,G.,Ward,W。W.和Prasher,D.C。(1994)。 绿色荧光蛋白作为基因表达的标记。 Science 263(5148):802-805。
  2. Mabuchi,K.,Maki,H.,Itaya,T.,Suzuki,T.,Nomoto,M.,Sakaoka,S.,Morikami,A.,Higashiyama,T.,Tada,Y.,Busch,W。 Tsukagoshi,H。(2018)。 MYB30将ROS信号,根细胞伸长和植物免疫反应联系起来。 Proc Natl Acad Sci USA 115(20):E4710-E4719。
  3. Nomoto,M。和Tada,Y。(2018a)。 使用多功能引物设计通过两步PCR进行无克隆蛋白质合成的无克隆模板DNA制备3'-UTR短。 Genes Cells 23(1):46-53。
  4. Nomoto,M。和Tada,Y。(2018b)。 植物转录因子的无细胞蛋白质合成。 方法MolBiol 1830:337-349。
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引用:Nomoto, M., Tada, Y. and Tsukagoshi, H. (2019). In vitro Protein-DNA Binding Assay (AlphaScreen® Technology). Bio-protocol 9(3): e3155. DOI: 10.21769/BioProtoc.3155.
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