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Mar 2019
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Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
重组输入蛋白-组蛋白配合物分子大小的超离心分析   

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Abstract

Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the mechanism of histone transport and also the potential roles of importins as histone chaperones and in the assembly of nucleosomes. Here we report a simple and reproducible sedimentation-velocity based method to determine the molecular sizes of importin-histone complexes using analytical ultracentrifugation. The method does not use any reporter tags or interaction with column resin thereby analyzing the interactions of the native proteins.

Keywords: Importin (输入蛋白), Histones (组蛋白), Sedimentation velocity (沉降速度), Molecular size (分子大小), Analytical ultracentrifugation (分析超离心)

Background

Nucleosomes are the most basic structural and functional units of the eukaryotic chromatin. Histone proteins H2A, H2B, H3 and H4 are the protein components of the nucleosomes. Each nucleosome consists of 147 base pairs of DNA wrapped around an H3-H4 tetramer and two copies of the H2A-H2B dimer (Luger et al., 1997a). Histones, like other proteins in the cell, are synthesized in the cytoplasm. The nucleosomes, however, are assembled in the nucleus. Despite their small size (monomers are 10-15 kDa), histones do not diffuse through the nuclear pore complex and instead are transported either alone or in complex with histone chaperones by the importins (Johnson-Saliba et al., 2000; Baake et al., 2001; Mosammaparast et al., 2001, 2002a and 2002b; Muhlhausser et al., 2001; Jakel et al., 2002).

Analysis of the histone-importin complexes helps elucidate the mechanism of the transport of histones. Histone proteins are highly basic proteins and have non-specific interactions with most column resins, and thus chromatography-based experiments require careful optimization and analyses. Here we describe an alternate solution-based sedimentation-velocity method to accurately determine the sedimentation coefficient of the various possible complexes. This Analytical Ultracentrifugation (AUC) method is highly reproducible and requires very little protein. Furthermore, it does not use any reporter tags, enabling experimentation with native or native-like macromolecules. The method can easily be adopted to study other importin-histone complexes and histone-chaperone complexes not only expressed in bacteria but also from other native sources.

Materials and Reagents

  1. Culture flask (1,000 ml) (VWR, catalog number: 29136-106 )
  2. Parafilm (Sigma-Aldrich, catalog number: BR701605 )
  3. FisherbrandTM Regenerated cellulose dialysis tubing (6,000-8,000D) (Fisher Scientific, catalog number: 21-152-4 )
  4. Slide-A-LyzerTM MINI Dialysis Device, 7K MWCO, 0.1 ml (Thermo Fisher Scientific, catalog number: 69560 )
  5. Glass Econo-Column (Bio-Rad, catalog number: 7374156 )
  6. Amicon Ultra-15 Centrifugal Filter Unit, 3kDa (Millipore Sigma, catalog number: UFC900324 )
  7. Amicon Ultra-15 Centrifugal Filter Unit,10kDa (Millipore Sigma, catalog number: UFC901024 )
  8. Amicon Ultra-15 Centrifugal Filter Unit, 50kDa (Millipore Sigma, catalog number: UFC905024 )
  9. E. coli BL21 DE3 plysS cells (Thermo Fisher Scientific, catalog number: C602003 )
  10. pET-3a plasmid (Sigma-Aldrich, catalog number: 69418 )
  11. pET-22b plasmid (Sigma-Aldrich, catalog number: 69744 )
  12. pGEX-4T3 plasmid (GE Healthcare, catalog number: 28954552 )
  13. Ampicillin (Goldbio, catalog number: A-301-5 )
  14. Quick start Bradford Protein Assay Kit (Bio-Rad, catalog number: 5000201 )
  15. Chloramphenicol (Goldbio, catalog number: C-105-5 )
  16. Complete, EDTA-free protease inhibitor (Sigma-Aldrich, catalog number: 11873580001 )
  17. Adenosine 5′-triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A26209 )
  18. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855 )
  19. DTT (RPI, catalog number: D110000-25.0 )
  20. EDTA (RPI, catalog number: E57020-500.0 )
  21. Glycerol (Fisher, catalog number: 633-4 )
  22. Tryptone (Sigma-Aldrich, catalog number: T7293 )
  23. Tris HCl pH 7.5 (Sigma-Aldrich, catalog number: T5941 )
  24. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  25. Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
  26. Magnesium acetate (Sigma-Aldrich, catalog number: M5661 )
  27. Imidazole (Sigma-Aldrich, catalog number: I5513 )
  28. Guanosine 5'-Triphosphate Sodium (Sigma-Aldrich, catalog number: G8877 )
  29. Glutathione sepharose 4B (GE Healthcare, catalog number: 17075601 )
  30. Guanidine HCl (RPI, catalog number: G49000-100
  31. HEPES (RPI, catalog number: H75030-500.0 )
  32. IPTG (Gold Biotechnology, catalog number: I24816100 )
  33. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  34. Ni-NTA Agarose (Qiagen, catalog number: 30320 )
  35. Sodium acetate (Sigma-Aldrich, catalog number: S8750 )
  36. Sodium chloride (NaCl) (RPI, catalog number: S23020-5000.0 )
  37. TCEP (Gold Biotechnology, catalog number: TCEP25 )
  38. Tris hydroxymethyl aminomethane (RPI, catalog number: T60040-5000.0 )
  39. Triton X-100 (Sigma-Aldrich, catalog number: T878-100ML )
  40. Urea (RPI, catalog number: U20200 )
  41. β-mercapto-ethanol (Sigma-Aldrich, catalog number: M6250 )
  42. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  43. LB media (1 L) (see Recipes)
  44. 2x YPT Media (1 L) (see Recipes)
  45. Wash buffer (see Recipes)
  46. Unfolding buffer (see Recipes)
  47. Refolding buffer (see Recipes)
  48. Sodium acetate urea buffer 200 (SAU 200) (see Recipes)
  49. Sodium acetate urea buffer 600 (SAU 600) (see Recipes)
  50. AUC buffer (see Recipes)
  51. Imp9-lysis buffer (see Recipes)
  52. Imp9-wash buffer (see Recipes)
  53. Sodium chloride Q buffer (see Recipes)
  54. Imp9-SEC buffer (see Recipes)
  55. Ran-lysis buffer (see Recipes)
  56. Ran-wash buffer (see Recipes)
  57. Ran-elution buffer (see Recipes)
  58. Sodium chloride SP buffer (see Recipes)
  59. Ran GTP exchange buffer (see Recipes)

Equipment

  1. Q-500 Sonicator (Q Sonica, catalog number: Q500-110 )
  2. Emulsiflex–C5 cell homogenizer (Avestin, catalog number: Emulsiflex C5 )
  3. Oakridge tubes–50 ml tubes (Thermo Fisher Scientific, catalog number: 3119-0050 )
  4. AKTA pure (chromatography system) (GE Healthcare, catalog number: 29046665 )
  5. HiTrap Q HP (GE Healthcare, catalog number: 17115301 )
  6. HiTrap SP HP (GE Healthcare, catalog number: 17115201 )
  7. Superdex 200 Increase 10/300 GL (GE Healthcare, catalog number: 28990944 )
  8. NanoDrop (Thermo Fisher Scientific, catalog number: ND-2000C )
  9. Lyophilizer (Labconco, catalog number: 7740020 )
  10. Avanti J-301 High-Performance Centrifuge (Beckman Coulter, catalog number: 363118 )
  11. JA-20 Beckman rotor (Beckman Coulter, catalog number: 334831 )
  12. Eight-hole An-50Ti rotor (Beckman Coulter, catalog number: 363782 )
  13. AUC (Analytical Ultracentrifuge) centerpiece assemblies, including charcoal-filled Epon centerpieces, sapphire windows, aluminum housings, and fill-port plugs (Beckman Coulter, catalog number: A37299 )
  14. Beckman-Coulter Optima XL-1 Analytical Ultracentrifuge (AUC) (Beckman Coulter, catalog number: B86437 )

Software

  1. SEDNTERP 1 (http://www.jphilo.mailway.com/download.htm)
  2. SEDFIT (http://www.analytical.ultracentrifugation.com)
  3. REDATE (http://biophysics.swmed.edu/MBR/software.html)
  4. GUSSI (http://biophysics.swmed.edu/MBR/software.html)

Procedure

Note: Histone purification and H2A-H2B complex assembly protocol is adapted from a previously published protocol (Luger et al., 1997b and 1999). The protocol has been altered slightly to suit the requirements of the current experiment. This protocol describes a methodology for analysis of Imp9–H2A-H2B complexes and can be used to study not only other Importin–histone complexes but also various histone chaperone complexes. A flow chart of the protocol is shown in Figure 1.


Figure 1. Flow chart depicting various steps of protein purification and analytical ultracentrifugation


  1. Expression of histones H2A/H2B
    1. Transform E. coli BL21 DE3 plysS cells with histone expression plasmid (pET-3a containing H2A/H2B gene) and plate on 2x YPT agar plates containing appropriate antibiotics (ampicillin and chloramphenicol). Incubate at 37 °C overnight (12-15 h). Fresh transformation will ensure better protein expression.
    2. Pick single colonies and start 3-5 ml pre-culture in 2x YPT media with appropriate antibiotic. Grow them at 37 °C overnight.
    3. Inoculate 100 ml 2x YPT media with antibiotics with 100 µl turbid pre-cultures and incubate until the OD reaches 0.4-0.5.
    4. Inoculate six 1 L 2x YPT media containing antibiotics evenly with the culture from Step A3 and grow at 37 °C until the OD reaches 0.4-0.5 and induce with 250 μM IPTG for 4 h at 37 °C.
    5. After 4 h, harvest the cells by centrifugation at 4,000 x g for 20 min at 4 °C.
    6. Resuspend the cell pellets in 35 ml/L (cell culture) of wash buffer.
    7. Flash freeze the cell suspension in liquid nitrogen and store at -80 °C. The frozen cell suspension can be stored for a week at -80 °C.

  2. Histone purification from inclusion bodies
    1. Thaw the cell suspension at room temperature (25 °C) and place the tube containing the cell suspension in an ice-beaker with the sonicator probe in the tube.
    2. Set up the sonicator on pulse mode (1 s on and 1 s off). Lyse the cells on pulse mode at 50% amplitude for 3 min, repeating it 3-4 times or until the cell suspension becomes less viscous. The pulse mode and ice-beaker help prevent over-heating of the sample.
    3. Transfer the lysate into Oakridge tubes and spin at 25,000 x g for 20 min at 4 °C. Discard the supernatant and resuspend the pellet with 35 ml/L (cell culture) of wash buffer containing 1% (v/v) Triton X-100.
    4. Spin at 25,000 x g for 20 min at 4 °C. Discard the wash buffer.
    5. Repeat the wash described above twice with wash buffer without Triton X-100. Discard the wash buffer after the spin (25,000 x g for 20 min at 4 °C). Collect the final pellet. The pellet contains the protein (H2A/H2B) inclusion bodies.
    6. The inclusion body pellet can be stored at -20 °C for a week.
    7. Thaw the inclusion body pellet at room temperature first by dissolving the pellet in 1ml of dimethyl sulfoxide (DMSO) and then suspending it well in 20 ml of unfolding buffer. Incubate it on a rocker gently (10-15 rpm) at room temperature for 30 min.
    8. Spin the suspension at 25,000 x g for 20 min at 4 °C to remove any particulates.
    9. Transfer the supernatant from the previous step carefully to the dialysis tubing (6-8 kDa cutoff).
    10. Dialyze the sample overnight in SAU-200 (4 x 1 L), changing the buffer every hour for the first three rounds and leaving the last one overnight. All dialysis steps are carried out at 4 °C.
    11. Carefully transfer the dialyzed sample to Oakridge tubes and spin at 25,000 x g for 20 min at 4 °C to remove any particulates.
    12. Connect HiTrap SP HP (Cation exchange chromatography) column to the FPLC system (AKTA pure) and equilibrate with SAU-200. Set up a FPLC program to inject the sample and run a step gradient from 0% SAU 600 (100% SAU 200) to 40% SAU 600 (60% SAU 200) in 10 column volumes and 40% SAU 600 (60% SAU 200) to 100% SAU 600 (0% SAU 200) in 10 column volumes. 200 ml each of SAU200 and SAU600 should be enough to form the step gradient in a 5 ml HiTrap SP HP column.
    13. Inject the dialyzed sample into HiTrap SP HP column pre-equilibrated with SAU-200.
    14. Elute histones bound to the HiTrap SP HP column using the step gradient setup in Step B12.
    15. H2A/H2B elutes at about 38-42% SAU 600. Pool the peak fractions from HiTrap SP HP column and transfer carefully to a dialysis tubing (6-8 kDa cutoff) and dialyze in ice-cold water (Water + 5 mM β-mercapto-ethanol) overnight (4 × 4 L), changing the water every hour for the first three rounds and leaving the last one overnight.
    16. Determine the concentration of histones in the dialyzed sample by measuring the absorbance at 280 nm against water using a NanoDrop and applying the known extinction coefficient and path length in Beer’s Law and aliquot the dialyzed samples into cryovials at approximately 1 mg total protein per aliquot. The theoretical molar extinction coefficients can be obtained using expasy protoparam tool (http://ca.expasy.org/tools/protparam.html).
    17. Prepare an icebox with dry ice and fill it with ice-cold ethanol.
    18. Transfer the cryovials from Step B16 into the icebox to flash freeze.
    19. Remove the cap of the cryovials and seal with parafilm and puncture the parafilm.
    20. Turn on the Lyophilizer and close the ballast. Wait for the vacuum to reach < 100 mT and condenser temperature to -40 °C.
    21. Load samples into the Lyophilizer bottles. Open the vacuum valve and let it run overnight.
    22. After the run, turn off the vacuum and take out the samples. Remove the parafilm and replace with cap.
    23. Store at -80 °C until ready to assemble the complex. Lyophilization extends the shelf life of histone proteins by several months.

  3. Histone dimer H2A-H2B complex assembly
    1. Dissolve lyophilized histone aliquots to a concentration of approximately 2 mg/ml in unfolding buffer and incubate at room temperature for at least 30 min but not more than one hour. Determine the concentration of unfolded histones by measuring the absorbance at 280 nm against the unfolding buffer using a NanoDrop and applying the known extinction coefficient and path length in Beer’s Law.
    2. Mix resuspended equimolar mixtures of H2A and H2B and dilute the mixture to 1 mg/ml in unfolding buffer and incubate them on a room temperature rocker at 10-15 rpm for 1 h.
    3. Centrifuge the sample at 25,000 x g for 10 min at 4 °C to remove any precipitates and transfer the supernatant to a dialysis tubing (6-8 kDa cutoff).
    4. Dialyze in refolding buffer overnight at 4 °C, changing the buffer at least four times (4 × 2 L), every hour for the first three rounds and leaving the last one overnight.
    5. Centrifuge the dialyzed sample at 25,000 x g for 10 min at 4 °C to remove any particulates and concentrate the sample in a 10 kDa Amicon centrifugal concentrator to an appropriate injection volume (A maximum load of 0.5 ml of 10 mg total protein is recommended per injection for good resolution on the S200 Superdex increase column).
    6. Inject the sample onto Superdex S200 increase column pre-equilibrated with refolding buffer. H2A-H2B dimer elutes at an elution volume of approximately 16.5 ml in a 24 ml Superdex S200 increase column.
    7. Pool the peak fractions, concentrate in a 10 kDa Amicon centrifugal concentrator to approximately 10 mg/ml.
    8. Aliquot into 100 μl aliquots, flash freeze and store them at -80 °C.

  4. Expression of Imp9
    1. Transform E. coli BL21 DE3 cells with GST-Imp9 expression plasmid (modified pGEX-4T3 (thrombin site replaced with TEV protease cleavage site [Chook and Blobel, 1999]) containing Imp9 gene) (Padavannil et al., 2019) and plate on LB agar plates containing ampicillin. Incubate at 37 °C overnight. Fresh transformation will ensure better protein expression.
    2. Pick single colonies and start 3-5 ml pre-culture in LB media with ampicillin. Grow them at 37 °C overnight.
    3. Inoculate 100 ml LB media with ampicillin with 0.5-1 ml turbid pre-cultures and incubate until the OD reaches 0.4-0.5.
    4. Inoculate 4 x 1 L LB media containing ampicillin evenly with the culture from Step D3 and grow at 37 °C until the OD reaches 0.6 and induce with 500 μM IPTG for 12 h at 20 °C.
    5. Harvest the cells by centrifugation at 4,000 x g for 20 min at 4 °C.

  5. Purification of Imp9
    1. Suspend the harvested cells in Imp9-lysis buffer.
    2. Lyse the cells in Emulsiflex–C5 cell homogenizer.
    3. Transfer the lysate into Oakridge tubes and spin at 40,000 x g for 30 min at 4 °C and collect the supernatant.
    4. Set up a gravity flow in a Glass Econo-Column column in the cold room (4 °C) and equilibrate 1.5 ml (per 1 liter of cell culture) of Glutathione Sepharose 4B resin with Imp9-lysis buffer.
    5. Elute out the equilibration lysis buffer from Step E4 and add the supernatant from Step E3. Pass the supernatant through the resin by gravity flow a couple of times.
    6. Wash the GST–Imp9 bound resin with the Imp9-wash buffer twice.
    7. Wash the GST–Imp9 bound resin with the Imp9-ATP wash buffer once (Imp9-ATP wash buffer is lysis buffer with 5 mM ATP).
    8. Wash the GST–Imp9 bound resin with an additional Imp9-wash buffer.
    9. Check the concentration (a rough estimate using Bradford reagent should suffice) of the fusion protein on beads to determine the amount of TEV protease to be added.
    10. Cleave GST tag on column by incubating GST–Imp9 bound resin with TEV protease containing Im9-wash buffer overnight at 4 °C (add 100 μl of TEV protease (100 μM) for every 50 mg of fusion protein). Gently mix it once and incubate at 4 °C. Do not rock the column after addition of TEV protease.
    11. Elute Imp9 from the column. GST tag stays bound to the resin.
    12. Connect HiTrap Q HP (Anion exchange chromatography) column to the FPLC system (AKTA pure) and equilibrate with 100 mM sodium chloride Q buffer. Set up an FPLC program to inject the sample and run a linear gradient from 100% 100 mM sodium chloride Q-buffer to 100% 1 M sodium chloride Q-buffer in 20 column volumes. 150 ml of each buffer should be enough to form the linear gradient in a 5 ml HiTrap Q HP column.
    13. Inject Imp9 from Step E11 to HiTrap Q HP column pre-equilibrated with 100 mM sodium chloride Q buffer.
    14. Elute the protein from the column using the set linear gradient of 100% 100 mM sodium chloride Q-buffer to 100% 1 M sodium chloride Q-buffer (Imp9 elutes at approximately 22% B [78% A and 22% B]. The peak is quite distinct and the fractions within the peak are pooled).
    15. Pool the fractions containing Imp9 and concentrate the protein using 50 kDa Amicon centrifugal concentrator to an appropriate injection volume (A maximum load of 0.5 ml of 10 mg total protein is recommended per injection for good resolution on the S200 Superdex increase column).
    16. Inject the protein from Step E15 to an S200 Superdex increase column pre-equilibrated with Size-exclusion buffer (Imp9-SEC buffer). Imp9 elutes at an elution volume of approximately 13 ml in a 24 ml column.
    17. Pool the peak fractions from the column and concentrate the protein using 50 kDa Amicon centrifugal concentrator to the required concentration (Imp9 can be concentrated to up to 20 mg/ml. The concentrated sample can be flash frozen in liquid nitrogen and stored at -80 °C until ready to use).

  6. Expression of Ran [yeast Gsp1 (1-179, Q71L)]
    1. Transform E. coli BL21 DE3 cells with Ran [Gsp1 (1-179, Q71L)] expression plasmid (pET-22b containing yeast Ran [Gsp1 (1-179, Q71L) gene] and plate on LB agar plates containing ampicillin. Incubate at 37 °C overnight. Fresh transformation will ensure better protein expression.
    2. Pick single colonies and start 3-5 ml pre-culture in LB media with ampicillin. Grow them at 37 °C overnight.
    3. Inoculate 100 ml LB media with ampicillin with 0.5-1 ml turbid pre-cultures and incubate until the OD reaches 0.4-0.5.
    4. Inoculate 4 x 1 liter LB media containing ampicillin evenly with the culture from Step F3 and grow at 37 °C until the OD reaches 0.6 and induce with 300 μM IPTG for 12 h at 20 °C. Ran expresses as Ran–His6. The His6-tag is not cleaved during purification.
    5. Harvest the cells by centrifugation at 4,000 x g for 20 min at 4 °C.

  7. Purification and GTP loading of Ran
    1. Suspend the harvested cells in Ran-lysis buffer.
    2. Lyse the cells in Emulsiflex–C5 cell homogenizer.
    3. Transfer the lysate into Oakridge tubes and spin at 40,000 x g for 30 min at 4 °C and collect the supernatant.
    4. Set up a gravity flow in a Glass Econo-Column column in the cold room (4 °C) and equilibrate 1.5 ml (per 1 liter of cell culture) of Ni-NTA agarose resin with Ran-lysis buffer.
    5. Elute out the equilibration lysis buffer from Step G4 and add the supernatant from Step G3. Pass the supernatant through the resin by gravity flow a couple of times.
    6. Wash the Ran bound resin with the Ran-wash buffer twice.
    7. Elute the Ran from the column with Ran-elution buffer.
    8. Load the eluted Ran with GTP by incubating it on ice for 30 min with 40 mM GTP (final concentration) (add GTP to required concentration from 100 mM GTP stock solution).
    9. Connect HiTrap SP HP (Cation exchange chromatography) column to the FPLC system (AKTA pure) and equilibrate with 50 mM sodium chloride SP-buffer. Set up an FPLC program to inject the sample and run a linear gradient from 100% 50 mM sodium chloride SP buffer to 100% 1 M sodium chloride SP buffer in 20 column volumes. 150 ml of each buffer should be enough to form the linear gradient in a 5 ml HiTrap SP HP column.
    10. Inject the GTP-loaded Ran to pre-equilibrated HiTrap SP HP column.
    11. Elute the protein using the set linear gradient of 100% of 50 mM sodium chloride SP-buffer to 100% 1 M sodium chloride SP-buffer [Ran elutes at 40% SP-buffer B (40% B and 60% A) The peak is quite distinct and the fractions within the peak are pooled].
    12. Pool the fractions containing Ran-GTP and concentrate the protein to 10 mg/ml using 3-kDa Amicon centrifugal concentrator. Aliquot the concentrated protein to 100 μl aliquots and store at -80 °C.

  8. Sample preparation for AUC
    1. Dialyze preassembled histones sequentially at 4 °C in 1 L of 1 M NaCl refolding buffer (2 h), 1 L of 500 mM NaCl refolding buffer (2 h), and 2 L of AUC buffer (overnight). Histone dimers tend to dissociate and aggregate on sudden exposure to low salt. Sequential dilution over time helps to maintain the dimer. Histone tetramers and histone octamer probably behave the same and should be treated similarly.
    2. Dialyze purified Imp9 at 4 °C in AUC buffer overnight.
    3. Dialyze purified Ran GTP at 4 °C in AUC buffer overnight.
    4. Inject the proteins (H2A-H2B dimer, Imp9 and Ran GTP separately) into Superdex S200 increase column pre-equilibrated with AUC buffer. Save the AUC buffer from the run to make dilutions and to use as a reference buffer in the AUC.
    5. Histone dimer (H2A-H2B) and Imp9 have an elution volume of 16 ml and 13 ml in a 24 ml Superdex S200 increase column respectively. Ran GTP elutes at an elution volume of 18 ml in a 24 ml Superdex S200 increase column. Pool the peak fractions and concentrate the proteins to approximately 10 mg/ml using Amicon centrifugal filter units. The proteins can be stored at -80 °C for a week. Also store the AUC buffer used to run the Superdex S200 column at -80 °C to avoid any buffer mismatch.

  9. Sample loading to the AUC cell
    1. Calculate the concentration of each protein required for the AUC run based on their molar extinction coefficients.
    2. Mix the dialyzed samples to the final volume of 450 μl for the sedimentation velocity experiment. 1) 450 μl Imp9 alone (3 μM), 2) 450 μl RanGTP alone (10 μM), 3) 450 μl H2A-H2B (10 μM), 4) 3 μM Imp9 + 3 μM RanGTP in a total volume of 450 μl, 5) 3 μM Imp9 +3 μM H2A-H2B in a total volume of 450 μl, 6) 3 μM Imp9 + 3 μM H2A-H2B + 10 μM RanGTP in a total volume of 450 μl. Incubate the proteins at 4 °C overnight the day before the AUC run to ensure proper equilibration of the complexes.
    3. Assemble standard Epon-filled centerpieces (Balbo et al., 2009) (Figure 2).


      Figure 2. Assembly of Epon-filled centerpieces. (a) An unassembled cell. The parts are (A) window liners, (B) window housings (with window cushions installed), (C) sapphire windows, (D) cell housing, (E) screw ring, (F) screw-ring gasket, (G) centerpiece, (H) fill-port plugs, and (I) fill-port gaskets. The window liners are placed into the window housings such that the gap in the liner is opposite the registration groove of the respective housing (seen at the top of both housings in this view. The windows are inserted into the window housings. One window is placed into the cell housing face up, then the centerpiece is inserted, then the second window (face down). On this, the screw-ring gasket is positioned, followed by the screw ring. The screw ring is torqued to between 120 and 140 in-lbs. The cell is filled with solutions through external fill ports, and then the fill port gaskets followed by the fill-port plugs are installed. Details are in Balbo et al. (2009). (b) The filled and assembled cell, viewed from the “top” of the cell.

    4. Set up the Beckman-Coulter Optima XL-1 Analytical Ultracentrifuge (AUC) for a sedimentation velocity experiment (Balbo et al., 2008).
    5. Load 450 μl of the samples into the sample sectors and load 450 μl of the reference buffer (AUC buffer) into the reference sectors of double-sector centerpieces and place them into an eight-hole An-50Ti rotor. Position the rotor in the centrifuge, and incubate under vacuum at 20 °C for 2.5 h. Commence centrifugation at 50,000 rpm.
      Monitor the sedimentation using absorbance at 280 nm (A280). Collect scans as rapidly as possible. Centrifugation may be ceased when all evidence of sedimentation is absent.

Data analysis

The end result of the data-analysis method detailed below is the c(s) distribution (Schuck, 2000). The result thus takes the form of a two-dimensional distribution, with the single population of species presented as a function of their respective sedimentation coefficients. Larger proteins or assemblies will have larger sedimentation coefficients. The method is based on the concept of scaling solutions to the Lamm Equation (Lamm, 1929) directly to the data (a(r,t)) according to



where r is the radius (in cm) from the center of rotation, s is the sedimentation coefficient, t is time in seconds since the beginning of the centrifugation, and D is the translational diffusion coefficient, and L depicts the Lamm Equation. This allows the AUC data to be directly fitted. Noise in the data can cause unrealistic, high-frequency fluctuations in the c(s) distribution, and thus it is regularized along lines discussed by Provencher (Provencher, 1982, Schuck, 2000, Schuck et al., 2002). Systematic noise elements in the data can easily be detected and removed, resulting in higher-quality fits (Schuck and Demeler, 1999). The sedimentation coefficients in the distributions, coupled with the refined frictional ratios from the analysis, can be used to determine molar masses for species and complexes, but in the latter case, these masses should only be relied on in situations when the complex is expected to be fully occupied for the entirety of the SV experiment and the refined frictional ratio can be safely assumed to represent that of the complex (i.e., most of the signal comes from the complex or all species detected have similar frictional ratios).
Note: Screen shots of various steps of data analysis are provided as a supplement to the article (Figure S1).

Sedimentation velocity data analysis

  1. Calculate the buffer density and viscosity from the buffer composition using SEDNTERP.
  2. Calculate the partial-specific volume of the protein using SEDNTERP (Laue et al., 1992) or SEDFIT (Zhao et al., 2011).
  3. Use REDATE to change the time stamps in the Beckman data using the algorithm suggested by the Schuck laboratory (Zhao et al., 2013).
    Note: REDATE optionally makes folders for each wavelength of data acquisition.
  4. Start SEDFIT
    1. Choose “Data → Load New Files”; load only data scans that show evidence of sedimentation, i.e., late scans with no evidence of sedimentation should be excluded. 50-150 scans are sufficient, and every “nth” scan may be loaded to observe this limit.
    2. Use the mouse to define the positions of the meniscus (red line), the sector bottom (blue line), and the data-analysis boundaries (green lines).
    3. Choose “Model → Continuous c(s) distribution” (Schuck, 2000).
    4. Choose “Parameters”:
      1. resolution 50/100
      2. s min 0
      3. s max 15/10
      4. refine (activate using the respective the check box)–frictional ratio (Proteins 1.2-2.0)
      5. refine–Baseline
      6. check–Fit time-independent noise
      7. check–Fit RI noise only if analyzing interferometric data
      8. refine–Meniscus
      9. confidence level (F-ratio)–set to 0.68 for 1 sigma of regularization.
      10. Input the partial-specific volume, solution density, and solution viscosity in their proper places.
  5. From the Main Menu, choose “Run” (i.e., refine all linear parameters).
  6. Adjust parameters if significant data/fit mismatches are in evidence. The most common culprits at this stage are frictional ratio and meniscus. Redo the Run until the fit lines reasonably resemble the data.
  7. From the Main Menu, choose “Fit” (i.e., iteratively refine all parameters).
  8. Assess the quality of the fit. The root-mean-square deviation (rmsd) should be low (usually less than 0.01 signal units), and minimal systematicity should be evidenced in the residual plots. Poor values/appearances at this stage may indicate data-acquisition problems, turbulence, or convection, and it may be necessary to redo the experiment if the data are so compromised. The default fitting option is the Simplex algorithm; change it to Marquardt Levenberg (“Options → Fitting Options → Marquardt Levenberg”), note the rmsd value, and fit the data again. Continue alternating between Simplex and Marquardt Levenberg until the rmsd values no longer change upon fitting.
  9. Under the conditions that (a) the species of interest dominates the signal or (b) all species may be assumed to have the same frictional ratio, the molar masses of species may be estimated by choosing “Display → Show peak Mw in c(s)” and pressing the button that appears in the c(s) distribution plot.
  10. Before plotting, it may be desirable to increase the resolution of the distribution (in the Parameters window) to 150 and redo the fit.
  11. Choose “Plot → GUSSI c(s) plot” for the first distribution. For subsequent distributions that are to be overlaid on the first, choose “Copy → Copy Distribution” (thereby placing it on the clipboard) and then paste it (“Distributions → Paste a Distribution”) into the GUSSI instance that contains previous distributions. A typical GUSSI output is shown in Figure 3.


    Figure 3. A typical GUSSI output. Analytical ultracentrifugation produced sedimentation profiles for Imp9, H2A-H2B, RanGTP, the 1:1 molar ratio mix of Imp9 and H2A-H2B dimer, the 1:1 molar ratio mix of Imp9 and RanGTP, and the 1:1:3 molar ratio mix of Imp9, H2A-H2B dimer and RanGTP.

  12. In GUSSI, choose “Integrate → Integrate All” to obtain weighted s-values for all species simultaneously (this can be done individually in SEDFIT as well by using the integration function, which is summoned by pressing Ctrl-I).
  13. Confidence intervals for weighted s-values (if necessary) (Schuck, 2016).
    1. In SEDFIT, after an optimized analysis, define the integration limits and note down the values.
    2. Note the optimized meniscus value.
    3. Choose “Statistics → Calculate variance ratio (F-statistics)” for a 68.3% confidence level and note down the target rmsd value.
    4. Choose “Parameters”: Fix the meniscus to a lower value (e.g., lower it by 0.01 cm) and perform a fit; observe the rmsd to see if it finishes above the target value. If it does not, lower the fixed meniscus value and repeat until the rmsd exceeds the target value.
    5. Keeping the meniscus fixed at this new value, choose “Statistics → Monte-Carlo for integrated weight → average s values” and perform a minimum of 1,000 iterations at a 68.3% confidence level.
    6. Note the confidence interval returned by the program (two values).
    7. Repeat, fixing the meniscus to values higher than the optimal one.
    8. Choose as the confidence interval the highest and lowest of the four values that were returned by the program.

Recipes

  1. LB media (1 L)
    10 g Tryptone
    10 g NaCl
    5 g Yeast extract
  2. 2x YPT Media (1 L)
    16 g Bacto Tryptone
    10 g Yeast extract
    5 g NaCl
  3. Wash buffer
    10 mM Tris HCl pH 7.5
    1 mM EDTA
    5 mM β-mercaptoethanol
  4. Unfolding buffer
    7 M Guanidinium HCl
    20 mM Tris HCl pH 7.5
    10 mM DTT
  5. Refolding buffer
    2 M NaCl
    10 mM Tris HCl
    1 mM EDTA
    5 mM β-mercaptoethanol
  6. Sodium acetate urea buffer 200 (SAU 200)
    7 M Urea
    20 mM Sodium Acetate, pH 5.2
    200 mM NaCl
    1 mM EDTA
    5 mM β-mercaptoethanol
  7. Sodium acetate urea buffer 600 (SAU 600)
    7 M Urea
    20 mM Sodium Acetate pH 5.2
    600 mM NaCl
    1 mM EDTA
    5 mM β-mercaptoethanol
  8. AUC buffer
    20 mM HEPES pH 7.3
    200 mM sodium chloride
    2 mM magnesium chloride
    2 mM TCEP
    8% glycerol
  9. Imp9-lysis buffer
    50 mM Tris-HCl pH 7.5
    100 mM NaCl
    1 mM EDTA
    2 mM DTT
    20% Glycerol
    Complete, EDTA-free protease inhibitor
  10. Imp9-wash buffer
    50 mM Tris-HCl pH 7.5
    100 mM NaCl
    1 mM EDTA
    2 mM DTT
    20% Glycerol
  11. Sodium chloride Q buffer
    20 mM Tris-HCl pH 7.5
    100 mM NaCl/1 M NaCl
    1 mM EDTA
    2 mM DTT
    20% Glycerol
  12. Imp9-SEC buffer
    20 mM HEPES pH 7.3
    110 mM potassium acetate
    2 mM magnesium acetate
    2 mM DTT
    15% Glycerol
  13. Ran-lysis buffer
    50 mM HEPES pH 8.0
    200 mM NaCl
    10% Glycerol
    2 mM magnesium acetate
    2 mM β-mercaptoethanol
    5 mM Imidazole
    Complete, EDTA-free protease inhibitor
  14. Ran-wash buffer
    20 mM HEPES pH 8.0
    200 mM NaCl
    10% Glycerol
    2 mM magnesium acetate
    2 mM β-mercaptoethanol
    40 mM imidazole
  15. Ran-elution buffer
    20 mM HEPES pH 7.5
    50 mM NaCl
    10% glycerol
    2 mM magnesium acetate
    2 mM β-mercaptoethanol
    300 mM imidazole
  16. Sodium chloride SP buffer
    20 mM HEPES pH 7.5
    50 mM NaCl/1 M NaCl
    4 mM magnesium acetate
    1 mM DTT
    10% glycerol
  17. Ran GTP exchange buffer
    20 mM HEPES pH 7.5
    100 mM NaCl
    4 mM magnesium acetate
    1 mM DTT
    10% glycerol

Note: The buffers are made using standard procedures. Weigh the components into a volume less than the final volume, adjust the pH while constantly stirring the buffer and after the pH has been adjusted, add water to achieve the final volume. Adjust the pH of the media to 7.0 before adding water to achieve the final volume.

Acknowledgments

We thank Bing Li for plasmids expressing H2A and H2B. This work was funded by NIGMS of NIH under Awards R01GM069909 (YMC), U01GM98256-01 (YMC), the Welch Foundation Grants I-1532 (YMC), the Leukemia and Lymphoma Society Scholar Award (YMC) and the University of Texas Southwestern Endowed Scholars Program (YMC). The histone purification and assembly protocol is a modified version of histone purification and assembly protocol from Karolin Luger’s published work.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Baake, M., Bauerle, M., Doenecke, D. and Albig, W. (2001). Core histones and linker histones are imported into the nucleus by different pathways. Eur J Cell Biol 80(11): 669-677.
  2. Balbo, A., Brown, P. H. and Schuck, P. (2008). Experimental Protocol for Sedimentation Velocity Analytical Ultracentrifugation.
  3. Balbo, A., Zhao, H., Brown, P. H. and Schuck, P. (2009). Assembly, loading, and alignment of an analytical ultracentrifuge sample cell. J Vis Exp(33). pii: 1530. doi: 10.3791/1530.
  4. Chook, Y. M. and Blobel, G. (1999). Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp. Nature 399(6733): 230-237.
  5. Jakel, S., Mingot, J. M., Schwarzmaier, P., Hartmann, E. and Gorlich, D. (2002). Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains. EMBO J 21(3): 377-386.
  6. Johnson-Saliba, M., Siddon, N. A., Clarkson, M. J., Tremethick, D. J. and Jans, D. A. (2000). Distinct importin recognition properties of histones and chromatin assembly factors. FEBS Lett 467(2-3): 169-174.
  7. Lamm, O. (1929). Die Differentialgleichung der Ultrazentrifugierung. Arkiv för matematik, astronomi och fysik 21B (2): 1-4.
  8. Laue, T., Shah, B. D., Rdigeway, R. M. and Pelletier, S. L. (1992). Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding, S. E., Rowe, A. J. and Horton, J. C. (Eds.). Analytical Ultracentrifugation in Biochemistry and Polymer Science. 90-125.
  9. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. and Richmond, T. J. (1997a). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648): 251-260.
  10. Luger, K., Rechsteiner, T. J., Flaus, A. J., Waye, M. M. and Richmond, T. J. (1997b). Characterization of nucleosome core particles containing histone proteins made in bacteria. J Mol Biol 272(3): 301-311.
  11. Luger, K., Rechsteiner, T. J. and Richmond, T. J. (1999). Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 119: 1-16.
  12. Mosammaparast, N., Ewart, C. S. and Pemberton, L. F. (2002a). A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B. EMBO J 21(23): 6527-6538.
  13. Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D. F. and Pemberton, L. F. (2002b). Pathways mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem 277(1): 862-868.
  14. Mosammaparast, N., Jackson, K. R., Guo, Y., Brame, C. J., Shabanowitz, J., Hunt, D. F. and Pemberton, L. F. (2001). Nuclear import of histone H2A and H2B is mediated by a network of karyopherins. J Cell Biol 153(2): 251-262.
  15. Muhlhausser, P., Muller, E. C., Otto, A. and Kutay, U. (2001). Multiple pathways contribute to nuclear import of core histones. EMBO Rep 2(8): 690-696.
  16. Padavannil, A., Sarkar, P., Kim, S. J., Cagatay, T., Jiou, J., Brautigam, C. A., Tomchick, D. R., Sali, A., D'Arcy, S. and Chook, Y. M. (2019). Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone. Elife 8: e43630.
  17. Provencher, S. W. (1982). CONTIN: A general purpose constrained regularization program for inverting noisy linerar algebraic and integral equations. Comput Phys Commun 27: 229-242.
  18. Schuck, P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78(3): 1606-1619.
  19. Schuck, P. (2016). Sedimentation Velocity Analytical Ultracentrifugation: Discrete Species and Size-Distributions for Macromolecules and Particles. ISBN: 9780367878283.
  20. Schuck, P. and Demeler, B. (1999). Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys J 76(4): 2288-2296.
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  22. Zhao, H., Brown, P. H. and Schuck, P. (2011). On the distribution of protein refractive index increments. Biophys J 100(9): 2309-2317.
  23. Zhao, H., Ghirlando, R., Piszczek, G., Curth, U., Brautigam, C. A. and Schuck, P. (2013). Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation. Anal Biochem 437(1): 104-108.

简介

[摘要] 组蛋白构成核小体的蛋白质成分。尽管其尺寸很小,但组蛋白不会通过核孔复合物扩散。取而代之的是,它们单独或与组蛋白分子伴侣复合地被重要蛋白转运至细胞核。确定importin-histone复合物的分子大小是理解组蛋白转运机制的关键,也是importins作为组蛋白伴侣和在核小体组装中的潜在作用的关键。在这里,我们报告了一种简单且可重现的沉降速度为基础的方法,该方法使用分析超速离心法来确定importin-histone配合物的分子大小。该方法不使用任何报告子标签或与色谱柱树脂的相互作用,从而分析了天然蛋白质的相互作用。

[背景] 核小体是真核染色质的最基本的结构和功能单元。组蛋白H2A,H2B,H3和H 4是核小体的蛋白质成分。每个核包括147个碱基的DNA wrapp的对编绕Ñ H3-H4四聚体和H2A-H2B二聚体的两个拷贝(Luger的等人,1997年一)。像细胞中的其他蛋白质一样,组蛋白在细胞质中合成。然而,核小体组装在核中。尽管它们的小尺寸(单体是10-15 kDa)的,组蛋白不通过核孔复合物扩散,而是可以单独使用或在复合物与由组蛋白importins伴侣输送要么(约翰逊-SAL IBA 等人,2000 ; Baake 等等人,2001;Mosammaparast 等人,2001,2002a和2002b;Muhlhausser 等人,2001;Jakel 等人,2002)。

组蛋白的分析- 输入蛋白复合物有助于阐明组蛋白的运输机制。组蛋白是高度碱性的蛋白质,并且与大多数柱状树脂具有非特异性相互作用,因此基于色谱的实验需要仔细的优化和分析。在这里,我们描述了另一种基于溶液的沉降速度方法,以准确确定各种可能的组分的沉降系数。这种分析超速离心(AUC)方法具有很高的重现性,并且只需要很少的蛋白质。此外,它不使用任何报告子标签,可以进行天然或类似天然大分子的实验。该方法可以很容易地用于研究不仅在细菌中表达而且从其天然来源表达的其他异丁香-组蛋白复合物和组蛋白-伴侣复合物。

关键字:输入蛋白, 组蛋白, 沉降速度, 分子大小, 分析超离心

材料和试剂


 


培养瓶(1 ,000毫升)(VWR,目录号:29136-106)
封口膜(Sigma-Aldrich,目录号:BR701605)
FISHERBRAND TM 再生纤维素透析管(6 ,000-8 ,000 d )(Fisher Scientific公司,目录号:21-152-4)
Slide-A-Lyzer TM MINI透析仪,7K MWCO,0.1 ml(Thermo Fisher Scientific,目录号:69560)
玻璃经济柱(伯乐,目录号:7374156)
Amicon Ultra-15离心过滤器单元,3kDa(Millipore Sigma,目录号:UFC900324)
Amicon Ultra-15离心过滤器单元,10kDa(Millipore Sigma,目录号:UFC901024)
Amicon Ultra-15离心过滤器单元,50kDa(Millipore Sigma,目录号:UFC905024)
E. 大肠杆菌BL21 DE3 pLysS细胞(赛默飞世尔科技,产品目录号:C602003)
pET-3a质粒(Sigma - Aldrich,目录号:69418)
pET-22b质粒(Sigma - Aldrich,目录号:69744)
pGEX-4T3质粒(GE Healthcare,目录号:28954552)
氨苄西林(Goldbio,目录号:A-301-5)
快速入门布拉德福德蛋白质测定试剂盒(Bio- R ad,目录号:5000201)
氯霉素(Goldbio,目录号:C-105-5)
完全,无EDTA的蛋白酶抑制剂(Sigma - Aldrich,目录号:11873580001)
腺苷5 ' 三磷酸二钠盐水合物(西格玛- Aldrich公司,目录号:A26209)
二甲基亚砜(DMSO)(Sigma - Aldrich,目录号:276855)
DTT(RPI,目录号:D110000-25.0)
EDTA(RPI,目录号:E57020-500.0)
甘油(Fisher,货号:633-4)
胰蛋白((Sigma-Aldrich,目录号:T7293)
Tris HCl pH 7.5 (Sigma-Aldrich,目录号:T5941)
β- 巯基乙醇(Sigma-Aldrich,目录号:M6250)
醋酸钾(Sigma-Aldrich,目录号:P1190)
醋酸镁(Sigma-Aldrich,目录号:M5661)
咪唑(Sigma-Aldrich,目录号:I5513)
鸟苷5'-三磷酸钠(Sigma - Aldrich,目录号:G8877 )
谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17075601)
盐酸胍(RPI,目录号: G49000-100 )
HEPES (RPI,目录号:H75030-500.0 )
IPTG(黄金生物技术,目录号:I24816100)
氯化镁(MgCl 2 )(Sig ma - Aldrich,目录号:M8266)
Ni-NTA琼脂糖(Qiagen,目录号:30320)
醋酸钠(Sigma - Aldrich,目录号:S8750)
氯化物钠È (氯化钠)(RPI,目录号:S23020-5000.0 )
TCEP(黄金生物技术,目录号:TCEP25)
Tris羟甲基氨基甲烷(RPI,目录号:T60040-5000.0)
的Triton X - 100(Sigma公司- Aldrich公司,目录号:T878 - 100ML)
尿素(RPI,货号:U20200)
β-巯基乙醇(Sigma - Aldrich,目录号:M6250)
酵母提取物(Sigma - Aldrich,目录号:Y1625)
LB介质(1 L)(请参阅食谱)
2x YPT介质(1 L)(请参阅食谱)
清洗缓冲液(请参阅配方)
展开缓冲区(请参见食谱)
重新折叠缓冲区(请参见食谱)
醋酸钠尿素缓冲液200(SAU 200)(请参阅食谱)
醋酸钠尿素缓冲液600(SAU 600)(请参阅配方)
AUC缓冲区(请参阅食谱)
Imp9裂解缓冲液(请参阅食谱)
Imp9清洗缓冲液(请参阅配方)
氯化钠Q缓冲液(请参阅食谱)
Imp9-SEC缓冲区(请参阅食谱)
溶血缓冲液(请参见配方)
冲洗缓冲液(请参见食谱)
Ran洗脱缓冲液(请参见配方)
氯化钠SP缓冲液(请参阅配方)
跑GTP 交换缓冲区(请参阅食谱)
 


设备


 


Q-500声波发生器(Q Sonica,目录号:Q500-110)
Emulsiflex– C5细胞匀浆器(Avestin,目录号:Emulsiflex C5)
Oakridge管– 50 ml管(Thermo Fisher Scientific,目录号:3119-0050)
AKTA pure(色谱系统)(GE Healthcare,目录号:29046665)
HiTrap Q HP(GE Healthcare,目录号:17115301)
HiTrap SP HP(GE Healthcare,目录号:17115201)
Superdex 200增加10/300 GL(GE Healthcare,目录号:28990944)
NanoDrop(Thermo Fisher Scientific,目录号:ND-2000C)
冻干机(Labconco,目录号:7740020)
阿凡提J-301高性能离心机(贝克曼Ç oulter,目录号:363118)
JA-20贝克曼转子(贝克曼C oulter,目录号334831)
八孔An-50Ti转子(Beckman C oulter,目录号:363782)
AUC(分析型超速离心机)核心组件,包括木炭填充的Epon中心焦点,蓝宝石窗口,aluminu 米外壳,和填充端口插头(贝克曼Ç oulter,目录号:A37299)
Beckman-Coulter Optima XL-1分析超速离心机(AUC)(Beckman C oulter,目录号:B86437 )
 


软件


 


SEDNTERP 1(http://www.jphilo.mailway.com/download.htm)
SEDFIT(http://www.analytical.ultracentrifugation.com)
REDATE(http://biophysics.swmed.edu/MBR/software.html)
GUSSI(http://biophysics.swmed.edu/MBR/software.html)
 


程序


 


注意:组蛋白纯化和H 2A-H2B复杂组装方案改编自先前公布的方案(Luger等,1997 b和1999 )。该协议已进行了略微更改,以适应当前实验的要求。Ť 他的协议描述的方法进行Imp9分析- H2A-H2B复合物,可用于研究不仅其他输入蛋白–组蛋白复合物,还有各种组蛋白伴侣复合物。该协议的流程图如图1所示。






D:\ Reformatting \ 2020-3-2 \ 1902971--1384 Abhilash Padavannil 755428 \ Figs jpg \图1.jpg


图1.气流C HART描绘各种步骤小号蛋白纯化和分析的一升超速离心


 


组蛋白H2A / H2B的表达
变换E. 大肠杆菌BL21 DE3 plysS中与组蛋白表达质粒的细胞(含有PET-3A H2A / H2B基因)和板对2X YPT琼脂含有合适的抗生素(氨苄青霉素和氯霉素)的板。在37孵育 ℃下过夜(12-15 1H) 。新鲜转化将确保更好的蛋白质表达。
挑选单个菌落,并在含有适当抗生素的2x YPT培养基中开始3-5 ml预培养。将它们在37 °C下生长过夜。
在100 ml 混浊的预培养物中接种100 ml 2x YPT培养基的抗生素,并孵育直至OD达到0.4-0.5。
用来自步骤A3的培养物均匀接种六种含有抗生素的1 L 2x YPT培养基,并在37°C下生长直至OD达到0.4-0.5,并在37°C下用250μMIPTG诱导4 h。
4之后小时,在4收获细胞,通过离心,000 X 克20分钟,在4 ℃。
将细胞沉淀重悬于35 ml / L的洗涤缓冲液(细胞培养液)中。
闪冻在液氮中,存储中的细胞悬浮液在- 80℃。将冷冻的细胞悬浮液可在贮存一周- 80℃。




从包涵体纯化组蛋白
在室温(25 °C)下解冻细胞悬液,然后将装有细胞悬液的试管放入冰杯中,并在其中放置超声探头。
将超声仪设置为脉冲模式(1秒钟打开和1秒钟关闭)。在50%振幅下以脉冲模式溶解细胞3分钟,重复3-4次或直到细胞悬液变粘稠为止。脉冲模式和烧杯有助于防止样品过热。
将裂解液转移到Oakridge管中,并在4 °C下以25,000 xg 旋转20分钟。弃去上清液,并用35 ml / L(细胞培养物)含有1%(v / v)Triton X-100的洗涤缓冲液重悬沉淀。
在4 °C下以25,000 xg 旋转20分钟。丢弃洗涤缓冲液。
用不含Triton X-100的洗涤缓冲液重复上述洗涤两次。旋转后丢弃洗涤缓冲液(在4 °C下25,000 xg,持续20分钟)。收集最后的沉淀。沉淀包含蛋白质(H2A / H2B)包涵体。
包涵体沉淀可以储存在- 20 ℃下一周。
首先在室温下解冻包涵体沉淀,方法是将沉淀溶解在1ml的二甲基亚砜(DMSO)中,然后将其充分悬浮在20 ml的展开缓冲液中。在室温下将其在摇杆上轻轻孵育(10-15 rpm)30分钟。
在4 °C下以25,000 xg 旋转悬浮液20分钟,以去除所有颗粒。
将上一步骤中的上清液小心地转移至透析管(截止6-8 kDa)。
在SAU-200(4 x 1 L)中将样品透析过夜,在前三轮中每小时更换一次缓冲液,而在最后一轮中过夜。所有透析步骤均在4 °C下进行。
小心地将渗析的样品转移到Oakridge管中,并在2 ° x 25,000 x g 下在4 °C 下旋转20分钟,以除去所有颗粒。
将HiTrap SP HP(阳离子交换色谱)色谱柱连接到FPLC系统(纯AKTA),并用SAU-200平衡。设置FPLC程序以进样并在10倍柱体积和40%SAU 600(60%SAU 200)中运行从0%SAU 600(100%SAU 200)到40%SAU 600(60 %SAU 200)的逐步梯度)至10柱体积的100%SAU 600(0 %SAU 20 0)。SAU200和SAU600各自200毫升应足以在5 毫升HiTrap SP HP色谱柱中形成阶梯梯度。
将透析后的样品注入用SAU-200预平衡的HiTrap SP HP色谱柱中。
洗脱组蛋白结合到使用在步骤梯度设置的SP的HiTrap HP柱小号TEP B12。
H2A / H2B的洗脱浓度约为SAU 600的38-42%。汇集HiTrap SP HP色谱柱的峰馏分,小心地转移至透析管(截止6-8 kDa),并在冰冷的水中透析(水+ 5 mMβ-巯基乙醇)过夜(4×4 L),在前三轮中每小时更换一次水,并在最后一轮中过夜。
通过使用NanoDrop 测量在280 nm处对水的吸光度并应用比尔定律中已知的消光系数和光程长度,确定透析样品中组蛋白的浓度,然后将透析后的样品分装到冷冻管中,每份等分试样的总蛋白量约为1 mg。可以使用实验原型工具(http://ca.expasy.org/tools/protparam.html)获得理论上的摩尔消光系数。  
准备一个有干冰的冰柜,然后装满冰冷的乙醇。
从转移的冷冻管小号TEP B16到冰箱闪烁冻结。
取下冷冻管的盖子,用封堵膜密封并穿刺封堵膜。
打开冻干机并关闭镇流器。等待真空访问< 100 MT和冷凝器温度至- 40 ℃。
将样品装入冻干瓶。打开真空阀,使其运行一整夜。
运行后,关闭真空并取出样品。取下封口膜并盖上盖子。
商店在- 80 ℃,直至准备组装复杂。冻干可将组蛋白的保存期限延长数月。
 


组蛋白二聚体H2A-H2B复合体
将冻干的组蛋白等分试样溶解在展开缓冲液中,使其浓度约为2 mg / ml,并在室温下孵育至少30分钟但不超过一小时。通过使用NanoDrop测量针对展开缓冲液在280 nm处的吸光度并应用比尔定律中已知的消光系数和光程长度,确定展开的组蛋白的浓度。
混合重悬的H2A和H2B等摩尔混合物,并在展开缓冲液中将其稀释至1 mg / ml,然后在室温摇床上以10-15 rpm孵育1小时。
在4 °C下以25,000 xg 离心样品10分钟,以除去所有沉淀物,并将上清液转移至透析管(截止6-8 kDa)。
在重新折叠的缓冲液中于4 °C 过夜透析,在前三轮中每小时更换缓冲液至少四次(4×2 L),最后一轮过夜。
在4 °C下以25,000 xg 离心透析的样品10分钟,以去除任何颗粒,然后在10 kDa Amicon离心浓缩器中将样品浓缩至适当的进样量(建议每次进样最大量为0.5 ml 10 mg总蛋白以获得在S200 Superdex增加色谱柱上的良好分离度)。
将样品注入Superdex S200增加柱中,并用重折叠缓冲液预平衡。H2A-H2B二聚体在24 ml Superdex S200增大柱中以大约16.5 ml的洗脱体积洗脱。
合并峰级分,在10 kDa Amicon离心浓缩器中浓缩至约10 mg / ml。
分装成100 μ升等分试样,闪存冻结并将其存储在-80 ℃。
 


Imp9的表达
变换E. 大肠杆菌BL21 DE3细胞用GST- Imp9表达质粒(改性的pGEX-4T3(凝血酶位与TEV取代蛋白酶切割位点[ Chook和布洛贝尔,1999 ] )含有Imp9 基因)(Padavannil 等人,2019)和板上含有氨苄青霉素的LB琼脂平板。在37 °C下孵育过夜。新鲜转化将确保更好的蛋白质表达。
挑选单个菌落,并在含有氨苄青霉素的LB培养基中开始3-5 ml预培养。将它们在37 °C下生长过夜。
用0.5-1 ml混浊的预培养物向100 ml LB培养基中加入氨苄青霉素,并孵育直至OD达到0.4-0.5。
用来自步骤D3的培养物均匀接种4 x 1 L 含氨苄青霉素的LB培养基,并在37°C下生长直至OD达到0.6,然后在20°C下用500μMIPTG诱导12 h。
收获通过离心将细胞在4 ,000 X 克20 分钟,4 ℃。
 


Imp9的纯化
将收获的细胞悬浮在Imp9裂解缓冲液中。
用Emulsiflex– C5细胞匀浆器裂解细胞。
将裂解物转移到Oakridge管中,并在40,000 x g 下于4°C 旋转30分钟,并收集上清液。
设置了在冷室(4玻璃Econo柱柱重力流动℃)和平衡1.5毫升(每1 升ITER细胞培养物)的谷胱甘肽琼脂糖4B树脂与Imp9裂解缓冲液中。
从S tep E4 洗脱平衡裂解缓冲液,并添加S tep E3 的上清液。通过重力流使上清液通过树脂几次。
用Imp9洗涤缓冲液洗涤GST –Imp9结合的树脂两次。
用Imp9-ATP洗涤缓冲液洗涤GST –Imp9结合的树脂一次(Imp9-ATP洗涤缓冲液是含5 mM ATP的裂解缓冲液)。
用附加的Imp9洗涤缓冲液洗涤与GST –Imp9结合的树脂。
检查珠子上融合蛋白的浓度(使用Bradford试剂进行粗略估计即可),以确定要添加的TEV蛋白酶的量。
上通过温育柱劈裂GST标签GST- Imp9结合的树脂与TEV蛋白酶含有IM9洗涤,在4缓冲液过夜℃(一个DD100μl的TEV蛋白酶(100μM)对每50毫克融合蛋白的)。轻轻混合一次并在4 °C 下孵育。加入TEV蛋白酶后,请勿摇动色谱柱。
从列中洗脱Imp9。GST标签保持与树脂的结合。
将HiTrap Q HP(阴离子交换色谱)色谱柱连接至FPLC系统(纯AKTA),并用100 mM 的氯化钠Q缓冲液平衡。设置一个Ñ FPLC程序注入样品和从100%运行的线性梯度的100mM 小号裂果氯化物Q-缓冲至100%1 中号小号在20个柱体积裂果氯化物Q-缓冲。每个缓冲液150 ml应该足以在5 ml HiTrap Q HP色谱柱中形成线性梯度。
注入Imp9从小号TEP E11到的HiTrap Q HP柱预平衡用100mM 小号裂果氯化物Q缓冲器。
使用100%100 mM s 氯化钠Q缓冲液到100%1 M s 氯化钠Q缓冲液的设定线性梯度从色谱柱上洗脱蛋白质(Imp9以大约22%B [ 78%A和22%B ] 洗脱峰非常明显,并且峰内的分数合并在一起。
合并含Imp9的馏分,并使用50 kDa Amicon离心浓缩器将蛋白质浓缩至合适的进样量(建议每次进样最大装载0.5 ml 10 mg总蛋白,以在S200 Superdex增加柱上获得良好的分离度)。
将蛋白从S tep E15 注入用大小排阻缓冲液(Imp9-SEC缓冲液)预先平衡的n S200 Superdex增加柱中。Imp9 在24 ml色谱柱中以大约13 ml 的洗脱体积洗脱。
收集色谱柱中的峰级分,并使用50 kDa Amicon离心浓缩仪将蛋白质浓缩至所需浓度(Imp9可以浓缩至20 mg / ml。浓缩的样品可以在液氮中快速冷冻并在-80 °C下保存C,直到可以使用为止)。  
 


Ran [ 酵母Gsp1(1-179,Q71L )]的表达
变换E. 大肠杆菌BL21 DE3细胞用冉[ GSP1(1-179,Q71L)] 表达质粒(含酵母冉的pET-22b的[ GSP1 (1-179,Q71L)基因] 和板在LB琼脂含有氨苄青霉素平板上。在温育37 °C过夜,新鲜转化可确保更好的蛋白质表达。
挑选单个菌落,并在含有氨苄青霉素的LB培养基中开始3-5 ml预培养。将它们在37 °C下生长过夜。
用0.5-1 ml混浊的预培养物向100 ml LB培养基中加入氨苄青霉素,并孵育直至OD达到0.4-0.5。
用来自步骤F3的培养物均匀接种4 x 1升含氨苄青霉素的LB培养基,并在37°C下生长直至OD达到0.6,然后在20°C下用300μMIPTG诱导12 h。Ran表示为Ran–His 6 。在纯化过程中,His 6 标签没有被切割。
收获通过离心将细胞在4 ,000 X 克20 分钟,4 ℃。
 


Ran的纯化和GTP负载
将收集的细胞悬浮在Ran裂解缓冲液中。
用Emulsiflex– C5细胞匀浆器裂解细胞。
将裂解物转移到Oakridge管中,并在40,000 x g 下于4°C 旋转30分钟,并收集上清液。
设置了在冷室(4玻璃Econo柱柱重力流动℃)和平衡1.5毫升(每1 升ITER细胞培养物)的Ni-NTA琼脂糖以冉裂解缓冲树脂。
              从S tep G4 洗脱平衡裂解缓冲液,并添加S tep G3 的上清液。通过重力流使上清液通过树脂几次。
用Ran洗涤缓冲液洗涤Ran结合的树脂两次。
用Ran洗脱缓冲液从色谱柱上洗脱Ran。
通过在冰上与40 mM GTP(最终浓度)一起孵育30分钟,将洗脱的Ran装载GTP(从100 mM GTP储备溶液中添加GTP至所需浓度)。
将HiTrap SP HP(阳离子交换色谱)色谱柱连接到FPLC系统(纯AKTA),并用50 mM 的氯化钠SP缓冲液平衡。设置n FPLC程序以进样,并在20倍柱体积中从100%50 mM s 氯化钠SP缓冲液到100%1 M s 氯化钠SP缓冲液运行线性梯度。每个缓冲液150 ml应该足以在5 ml HiTrap SP HP色谱柱中形成线性梯度。
将载有GTP的Ran注入预先平衡的HiTrap SP HP色谱柱。
使用100%的50 mM s 氯化钠SP缓冲液到100%1 M s 氯化钠SP缓冲液的设定线性梯度洗脱蛋白质[ Ran以40%SP缓冲液B(40%B和60%A)洗脱峰非常明显,峰内的馏分汇集在一起。
合并含有Ran-GTP的馏分,并使用3-kDa Amicon离心浓缩器将蛋白质浓缩至10 mg / ml 。等分试样浓缩的蛋白质至100μl等分试样并储存一吨-80℃ 。
 


AUC的样品制备
在1升1 M NaCl复性缓冲液(2 h),1升500 mM NaCl复性缓冲液(2 h)和2升AUC缓冲液(过夜)中于4°C 依次透析预组装的组蛋白。组蛋白二聚体倾向于在突然暴露于低盐下时解离和聚集。随时间顺序稀释有助于维持二聚体。组蛋白四聚体和组蛋白八聚体的行为可能相同,应进行类似处理。
在AUC缓冲液中于4 °C 过夜透析纯化的Imp9 。
在AUC缓冲液中于4 °C 透析纯化的Ran GTP 过夜。
将蛋白质(分别为H2A-H2B二聚体,Imp9和Ran GTP)注入Superdex S200增加柱中,并用AUC缓冲液预先平衡。从运行中保存AUC缓冲液以进行稀释并用作AUC中的参考缓冲液。
组蛋白二聚体(H2A-H2B)和Imp9在24 ml Superdex S200增加柱中的洗脱体积分别为16 ml和13 ml。Ran GTP在24 ml Superdex S200增加柱中以18 ml洗脱体积洗脱。合并峰级分,并使用Amicon离心过滤器将蛋白质浓缩至约10 mg / ml。这些蛋白质可以在-80 °C 下保存一周。还要在-80 °C 下存储用于运行Superdex S200色谱柱的AUC缓冲液,以避免任何缓冲液不匹配。
 


样品加载到AUC池
根据其摩尔消光系数,计算AUC运行所需的每种蛋白质的浓度。
混合透析样品至最终体积为450μl ,以进行沉降速度实验。1)450 微升单独Imp9(3 μ M),2)450 微升RanGTP单独(10 μ M),3)450 微升H2A-H2B(10 μ M),4)3 μ 中号Imp9 + 3 μ 中号RanGTP在450的总体积微升,5)3 μ 中号Imp9 3 μ 中号H2A-H2B在450的总体积微升,6)3 μ 中号Imp9 + 3 μ 中号H2A-H2B + 10 μ 中号RanGTP在总体积450 微升。在AUC运行前一天,将蛋白质在4 °C下孵育过夜,以确保复合物正确平衡。
组装标准的Epon填充中心配件(Balbo 等,2009)(图2)。
 


D:\ Reformatting \ 2020-3-2 \ 1902971--1384 Abhilash Padavannil 755428 \ Figs jpg \图2.jpg


图2. Epon填充中心件的组装。(a)未组装的牢房。零件包括(A)窗衬,(B )窗壳(已安装窗垫),(C)蓝宝石窗,(D)电池壳,(E)螺丝环,(F)螺丝环垫片,(G)中心件,(H)填充口塞和(I)填充口垫片。窗口衬垫放入窗口壳体,使得在所述衬垫的间隙为相应的壳体(在顶部看到的登记槽相反在此视图中两个壳体的。窗户是插件erted到窗口的壳体中。其中一个窗口是将电池盖正面朝上放置,然后插入中心件,然后插入第二个窗口(正面朝下),在该位置上,拧紧螺丝环垫圈,然后拧紧螺丝环,拧紧螺丝环的扭矩在120至140英寸·磅。该电池通过外部填充口填充有溶液,然后将填充口密封垫随后加料口塞是我nstalled。详细AR E在巴尔博等人。(2009)。(b)在填充从电池的“顶部”看,它是组装好的电池。


 


设置用于沉淀速度实验的Beckman-Coulter Optima XL-1分析型超速离心机(AUC)(Balbo et al。,2008)。
将450μl的样品装入样品扇区,并将450μl的参考缓冲液(AUC缓冲区)装入双扇区中心件的参考扇区,并将其放入八孔An-50Ti转子中。将转子置于离心机中,并在真空中于20 °C 孵育2.5 h 。在50开始离心,000转。
使用280 nm(A 280 )的吸光度监控沉降。尽快收集扫描。如果没有所有沉淀迹象,则可以停止离心。






数据分析


 


下面详细介绍的数据分析方法的最终结果是c (s )分布(Schuck ,2000年)。因此,结果采取二维分布的形式,物种的单一种群根据其相应的沉降系数呈现。较大的蛋白质或装配体将具有较大的沉降系数s。该方法基于将Lamm方程(Lamm ,1929)的解直接缩放为数据(a (r,t ))的概念。


 






 


其中r 是距旋转中心的半径(以厘米为单位),s 是沉降系数,t 是自离心开始以来的时间(以秒为单位),D 是平移扩散系数,L 表示拉姆方程。这样可以直接拟合AUC 数据。数据中的噪声会导致c (s )分布中不切实际的高频波动,因此它按照Provencher (Provencher ,1982 ; Schuck ,2000 ; Schuck et al。,2002)讨论的思路进行了正则化。数据中的系统噪声元素可以很容易地被检测和去除,从而获得更高质量的拟合度(Schuck and Demeler ,1999)。分布中的沉降系数,再加上分析中精确的摩擦比,可用于确定物质和配合物的摩尔质量,但在后一种情况下,仅应在预期配合物存在的情况下使用这些质量。在整个SV实验中完全被占用,并且可以安全地假定精细的摩擦比代表复合物的摩擦比(即,大多数信号来自复合物,或者检测到的所有物质都具有相似的摩擦比)。


注意:作为文章的补充,提供了数据分析各个步骤的屏幕快照(图S1)。


 


沉降速度数据分析


使用SEDNTERP从缓冲液组合物中计算缓冲液密度和粘度。
使用SEDNTERP (Laue 等,1992 )或SEDFIT (Zhao 等,2011)计算蛋白质的部分比容。
使用Schuck实验室(Zhao 等,2013)建议的算法,使用REDATE更改Beckman数据中的时间戳。
注意:REDATE可以选择为每个波长的数据采集创建文件夹。


启动SEDFIT
一个。选择“DAT 一→ 加载新文件”; 仅加载显示沉降迹象的数据扫描,即应排除没有沉降迹象的后期扫描。50-150次扫描就足够了,每次“第n次”扫描都可以加载以遵守此限制。       


b。使用鼠标定义弯月面(红线),扇区底部(蓝线)和数据分析边界(绿线)的位置。      


C。选择“模型→ 孔蒂nuous C(S)分配” (舒克,2000)。       


d。选择“参数” :      


[R esolution 50/100
小号分钟0
ŝ 最大15/10
ř efine(激活全光照克相应的复选框) - 摩擦比(蛋白质1.2-2.0)
[R efine- 基线
c heck – 适合时间- 独立噪音
入住飞度RI ň 瓦兹只有分析干涉数据
提炼– 半月板
Ç onfidence级(F-比) - 设定在0.68进行1-Σ正规化。
我NPUT特定部分卷,溶液密度,并在其适当的位置的溶液粘度。
从主菜单中选择“运行”(即,优化所有线性参数)。
如果明显的数据/拟合不匹配,请调整参数。在此阶段最常见的问题是摩擦比和弯液面。重做运行,直到拟合线合理地类似于数据为止。
从主菜单中,选择“ Fit”(即迭代优化所有参数)。
评估合适的质量。均方根偏差(rmsd)应该低(通常小于0.01信号单位),并且在残差图中应证明最小的系统性。此阶段的值/外观差可能表示数据获取问题,湍流或对流,如果数据受到损害,则可能有必要重新做实验。默认的拟合选项是Simplex算法;将其更改为Marquardt Levenbe rg(“选项→拟合选项→ Marquardt Levenberg”),记下rmsd值,然后再次拟合数据。继续在Simplex和Marquardt Levenberg之间交替,直到rmsd值在拟合后不再改变为止。
在以下条件下:(a)感兴趣的物种占主导地位,或(b)所有物种的摩擦比均假定为相同,可以通过选择“显示→ 在c(s )”,然后按c(s)分布图中出现的按钮。
在绘制之前,可能需要将分布的分辨率(在“参数”窗口中)提高到150,然后重新拟合。
对于第一个分布,选择“图→ GUSSI c(s)图”。对于将要覆盖的后续发行版,选择“复制→ 复制发行版”(从而将其放置在剪贴板上),然后将其粘贴(“发行版→ 粘贴发行版”)到包含先前发行版的GUSSI实例中。典型的GUSSI输出如图3所示。
 


D:\ Reformatting \ 2020-3-2 \ 1902971--1384 Abhilash Padavannil 755428 \ Figs jpg \图3.jpg


图3 。典型的GUSSI输出。分析型超速离心产生了Imp9,H2A-H2B,RanGTP,Imp9和H2A-H2B二聚体的1:1摩尔比混合物,Imp9和RanGTP的1:1摩尔比混合物以及1:1:3摩尔比的沉降曲线Imp9,H2A-H2B二聚体和RanGTP的混合物。


 


在GUSSI中,选择“积分→ 全部积分”以同时获得所有物种的加权s 值(这也可以通过使用Ctrl-I求和的积分函数在SEDFIT中单独完成)。
加权s 值的置信区间(如有必要)(Schuck ,2016年)。
一个。在SEDFIT中,经过优化分析后,定义积分极限并记下值。       


b。注意优化的弯液面值。      


C。为68.3%的置信度选择“统计信息→ 计算方差比(F统计量)”,并记下目标均方根值。       


d。选择“参数”:将弯液面固定为一个较低的值(例如,将其降低0.01厘米)并进行拟合;观察均方根值,看其是否超过目标值。如果不是,请降低固定的弯液面值,并重复进行,直到均方根值超过目标值。      


e。保持固定在这个新的价值半月板中,选择“统计→ 蒙特卡洛的整合重→ 平均价值观”,并执行最小为1 ,在68.3%的置信水平000迭代。       


F。注意程序返回的置信区间(两个值)。        


G。重复,将弯液面固定为高于最佳弯液面的值。      


H。选择程序返回的四个值中的最高值和最低值作为置信区间。      


 


菜谱


 


LB介质(1升)
10克胰蛋白Try


10克氯化钠


5克酵母提取物


2x YPT介质(1 L)
16克Bacto胰蛋白p


10克酵母提取物


5克氯化钠


洗涤缓冲液
10 mM Tris HCl pH 7.5


1毫米EDTA


5mM的β 巯基乙醇


展开缓冲
7 M盐酸胍


20 mM Tris HCl pH 7.5


10毫米DTT


重新折叠缓冲区
2 M氯化钠


10毫米Tris HCl


1毫米EDTA


5mM的β 巯基乙醇


乙酸钠尿素缓冲液200(SAU 200)
尿素7 M


20 mM醋酸钠,pH 5.2


200毫米氯化钠


1毫米EDTA


5mM的β 巯基乙醇


醋酸钠尿素缓冲液600(SAU 600)
尿素7 M


20 mM醋酸钠pH 5.2


600毫米氯化钠


1毫米EDTA


5mM的β 巯基乙醇


AUC缓冲区
20 mM HEPES pH 7.3


200米中号小号裂果Ç hloride


2mM的米agnesium Ç hloride


2毫米TCEP


8%甘油


Imp9裂解缓冲液
50 mM的Tris-HCl pH 7.5


100毫米氯化钠


1毫米EDTA


2毫米DTT


20%甘油


完全,不含EDTA的蛋白酶抑制剂


Imp9清洗缓冲液
50 mM的Tris-HCl pH 7.5


100毫米氯化钠


1毫米EDTA


2毫米DTT


20%甘油


氯化钠Q缓冲液
20毫米Tris-HCl pH 7.5


100 mM氯化钠/ 1 M氯化钠


1毫米EDTA


2毫米DTT


20%甘油


Imp9-SEC缓冲区
20 mM HEPES pH 7.3


110 mM的p otassium乙


2mM的米agnesium乙


2毫米DTT


15%甘油


裂解缓冲液
50 mM HEPES pH 8.0


200毫米氯化钠


10%甘油


2mM的米agnesium 一个cetate


2 mMβ-巯基乙醇


5 mM咪唑


完全,不含EDTA的蛋白酶抑制剂


冲洗缓冲液
20 mM HEPES pH 8.0


200毫米氯化钠


10%甘油


2mM的米agnesium 一个cetate


2 mMβ-巯基乙醇


40mM的我咪唑的


洗脱洗脱缓冲液
20 mM HEPES pH 7.5


50毫米氯化钠


10%克lycerol


2mM的米agnesium 一个cetate


2 mMβ-巯基乙醇


300mM的我咪唑的


氯化钠SP缓冲液
20 mM HEPES pH 7.5


50 mM氯化钠/ 1 M氯化钠


4毫米agnesium 一个cetate


1毫米DTT


10%克lycerol


跑GTP交换缓冲区
20 mM HEPES pH 7.5


100毫米氯化钠


4毫米agnesium 一个cetate


1毫米DTT


10%克lycerol


 


注意:使用标准程序制作缓冲区。称量组分至小于最终体积的体积,在不断搅拌缓冲液的同时调节pH值,调节完pH值后,加水达到最终体积。在加水以达到最终体积之前,将介质的pH调节至7.0。


 


致谢


 


我们感谢李兵表达H2A和H2B的质粒。这项工作由美国国立卫生研究院的NIGMS资助,奖项包括R01GM069909(YMC),U01GM98256-01(YMC),Welch Foundation I-1532(YMC),白血病和淋巴瘤协会学者奖(YMC)以及德克萨斯大学西南捐赠基金学者计划(YMC)。组蛋白纯化和装配规程是Karolin Luger出版的著作中组蛋白纯化和装配规程的修改版本。


 


利益争夺


 


作者声明没有利益冲突或利益冲突。






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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Padavannil, A., Brautigam, C. A. and Chook, Y. M. (2020). Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation. Bio-protocol 10(10): e3625. DOI: 10.21769/BioProtoc.3625.
  2. Padavannil, A., Sarkar, P., Kim, S. J., Cagatay, T., Jiou, J., Brautigam, C. A., Tomchick, D. R., Sali, A., D'Arcy, S. and Chook, Y. M. (2019). Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone. Elife 8: e43630.
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