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Sep 2017

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In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
古生菌中泛素样蛋白修饰的体外分析   

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Abstract

The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.

Keywords: Archaea (古生菌), Ubiquitin (Ub) (泛素(Ub)), Post-translational modification (翻译后修饰), SAMP (SAMP)

Background

The process by which ubiquitin (Ub) is covalently attached to target proteins is termed ubiquitination, which controls an enormous range of cellular process in eukaryotic cells (Glickman and Ciechanover, 2002; Komander and Rape, 2012). Ubiquitination is catalyzed by a cascade of enzymes including an Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and Ub ligases (E3s). In vitro reconstitution of ubiquitination is a useful assay to determine the specificity between enzymes or between E3s and protein substrates (Zhao et al., 2012). In Archaea, the Ubl protein SAMP adopts a Ub-fold and is isopeptide-linked to protein targets catalyzed by an E1-like enzyme UbaA [reviewed in Maupin-Furlow, (2014)]. While E1 homologs are widespread in Archaea, canonical E2 or E3 enzymes are not predicted in most Archaea based on primary sequence comparisons. Our recent study of Haloferax volcanii, shows methionine sulfoxide reductase A (MsrA) is needed for Ubl protein modification (sampylation) together with UbaA under a mild oxidative condition in vivo and in vitro (Fu et al., 2017). Here, we describe a detailed in vitro protocol to reconstitute and analyze MsrA-dependent sampylation.

Materials and Reagents

  1. Gloves (Fisherbrand, Fisher Scientific, catalog number: 19-130-1597C )
  2. Sterile toothpicks (Royal Paper Products, Inc., item number: R820)
  3. Ampac 500 series SealPAK heavy duty pouches (10.2 x 15.2 cm) (Fisher Scientific, catalog number: 01-812-25D) (seal Western blot membrane with CDP-Star or ECL prime reagents in pouches using colored labeling tape)
    Manufacturer: Ampac, catalog number: 50024 .
  4. Sterile loop (Fisherbrand, Fisher Scientific, catalog number: 22-363-599 )
  5. Fernbach flasks (2.8 L, wide mouth) (Corning, PYREX®, catalog number: 4420-2XL )
  6. Baffled culture flasks (250 ml) (DWK Life Sciences, Kimble, catalog number: 25630-250 )
  7. Aluminum foil (Fisherbrand, Fisher Scientific, catalog number: 01-213-105 ) (used to wrap items for autoclaving)
  8. Sterile polystyrene disposable serological 10 ml pipets with magnifier stripe (Fisherbrand, Fisher Scientific, catalog number: 13-678-11E )
  9. Polypropylene round-bottom centrifuge tubes, 50 ml (Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 , item number: UX-06327-47)
  10. Nalgene rapid-flow sterile disposable bottle top 0.2 µm filters with surfactant-free cellulose acetate (SFCA) membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 290-3320 )
  11. HisTrap HP column (5 ml) (GE Healthcare, catalog number: 17524802 )
  12. StrepTrap HP column (1 ml) (GE Healthcare, catalog number: 29048653 )
  13. 13 x 100 mm2 culture tubes (Fisher Scientific, catalog number: 14-961-27 )
  14. Borosilicate glass tubes with plain end (13 x 100 mm) (Fisherbrand, Fisher Scientific, catalog number: 14-961-27 ) (used for cell culture)
  15. Amicon Ultra-4 centrifugal filter unit with Ultracel-3 membrane (NMWL 3 kDa) (Merck, catalog number: UFC800308 )
  16. Superdex 75 10/300 GL column (GE Healthcare, catalog number: 17517401 )
  17. Surfactant-free cellulose acetate (SFCA) membrane syringe filters (28-mm membrane with 0.2, 0.45 and 0.8 µm pore sizes in acrylic housing) (Corning, catalog numbers: 431219 , 431220 , and 431221 )
  18. SnakeSkin dialysis tubing (3.5K MWCO, 22 mm I.D.) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 68035 )
  19. 1.5 ml microcentrifuge tubes (Fisherbrand, Fisher Scientific, catalog number: 02-681-320 )
  20. 2.0 ml microcentrifuge tubes (Fisherbrand, Fisher Scientific, catalog number: 02-681-321 )
  21. Zeba Spin Desalting Columns (7K MWCO) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 89882 )
  22. Amersham Hybond P 0.45 polyvinylidene difluoride (PVDF) membrane (GE Healthcare, catalog number: 10600023 )
  23. Disposable plastic cuvettes (Fisherbrand, Fisher Scientific, catalog number: 149-551-27 )
  24. X-ray film (RPI, catalog number: 248300 )
  25. Rack LTS tips (‘P20’ 2-20 µl, ‘P200’ 20-200 µl, ‘P1000’ 100-1,000 µl) (Mettler-Toledo, Rainin, catalog numbers: 17001865 , 17001863 and 17001864 )
  26. 10 ml Luer-Lok syringe (BD, catalog number: 309695 )
  27. Kimwipes Delicate Task Wipers, 1-Ply (KWCC, Kimberly-Clark, catalog number: 34155 )
  28. Colored labeling tape, rainbow pack (Fisherbrand, Fisher Scientific, catalog number: 15-901-10R )
  29. Four square cassettes (8 x 10") (Fisher Scientific, catalog number: FBXC-810 ) (for exposure of Western blot membrane to X-ray film)
  30. Glass storage/media bottles (DWK Life Sciences, Kimble, catalog numbers: 14395-500 for 500 ml and 14395-1000 for 1,000 ml)
  31. Polypropylene griffin low-form plastic beakers (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 1201-4000 for 2,000 ml and 1201-1000 for 1,000 ml)
  32. Polyvinyl wrapping film (Fisherbrand, Fisher Scientific, catalog number: 15-610 ) (can store SDS-PAGE gels wrapped in Kimwipes that are moistened with deionized water and further wrapped with polyvinyl film for up to 2 weeks at 4 °C prior to electrophoresis)
  33. Polystyrene cuvettes (1.5 ml capacity) (Fisherbrand, Fisher Scientific, catalog number: 14-955-127 ) (used for assays in visible spectral range, 340 to 750 nm)
  34. Polypropylene plastic graduated cylinders (Nalgene, Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 3664-0050 , 3664-0250 , and 3664-1000 )
  35. Polypropylene closures (DWK Life Sciences, Kimble, catalog number: 73660-13 ) (used with 13 mm O.D. plain-end culture tubes)
  36. Petri dishes with clear lid (Fisherbrand, Fisher Scientific, catalog number: FB0875712 )
  37. Cells
    1. Haloferax volcanii LR03 (Δsamp1 Δsamp2 Δsamp3 ΔmsrA ΔubaA) carrying plasmid pJAM3010 (a plasmid encoding msrA-strepII under control of the P2rrnA constitutive promoter) (Fu et al., 2017) or pJAM1209 (a plasmid encoding his6-ubaA under control of the P2rrnA constitutive promoter) (Hepowit et al., 2016) or without carrying any plasmid
      Note: H. volcanii strains and plasmids are available upon request.
    2. Escherichia coli Rosetta (DE3) (Novagen, Merck, catalog number: 70954-3 ) carrying plasmid pJAM3200 (a plasmid encoding msrA-strepII under control of T7 promoter) from the Maupin-Furlow lab (Fu et al., 2017) or plasmid pJAM1132 (a plasmid encoding flag-his6-samp2 under control of T7 promoter) from the Maupin-Furlow lab (Hepowit et al., 2016)
      Note: Plasmids are available from the Maupin-Furlow lab upon request.
  38. MsrA-StrepII (purified as per procedure outlined below)
  39. His6-UbaA (purified as per procedure outlined below)
  40. Deionized H2O (for details see Equipment item number 27)
  41. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  42. Novobiocin (Sigma-Aldrich, catalog number: N1628 )
  43. Kanamycin sulfate (Fisher BioReagents, Fisher Scientific, catalog number: BP906-5 )
  44. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fisher BioReagents, Fisher Scientific, catalog number: BP1620-1 )
  45. d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411-500MG )
  46. Imidazole, ACS Reagent, ≥ 99% titration (Sigma-Aldrich, catalog number: I2399 )
  47. Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, catalog number: 1610436 )
  48. Pierce Bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  49. Albumin, bovine (BSA) (lyophilized powder, ≥ 96% purity by agarose gel electrophoresis) (Sigma-Aldrich, catalog number: A2153 )
  50. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Chemical, Fisher Scientific, catalog number: M35-12 )
  51. Bortezomib proteasome inhibitor (free base, > 99% purity) (LC Laboratories, catalog number: B-1408 )
  52. DNase I from bovine pancreas (Sigma-Aldrich, catalog number: D4263-1VL ), a standardized vial containing 2,000 Kunitz units of DNase I (Sigma-Aldrich, catalog number: D4527 ) at ≥ 0.25 mg total protein
  53. Dimethylformamide (DMF) (Fisher BioReagents, Fisher Scientific, catalog number: BP1160-500 )
  54. Sodium chloride (NaCl) (Fisher Chemical, Fisher Scientific, catalog number: S642-12 )
  55. Adenosine 5’-triphosphate (ATP), disodium salt hydrate, 98% (ACROS Organics, catalog number: 102800100 )
  56. Dimethyl sulfoxide (DMSO), molecular biology grade (Sigma-Aldrich, catalog number: D8418-50ML ) (for in vitro assays)
  57. DMSO, Certified ACS (Fisher Chemical, Fisher Scientific, catalog number: D128-1 ) (for culturing cells)
  58. Dithiothreitol (DTT) (Fisher BioReagents, Fisher Scientific, catalog number: BP172-5 )
  59. Antibodies
    1. Strep-tag II monoclonal antibody (in mouse) (QIAGEN, catalog number: 34850 )
    2. Anti-His IgG2 monoclonal antibody (in mouse) (GE Healthcare, catalog number: 27471001 )
    3. HRP-conjugated His6 monoclonal antibody (Proteintech, catalog number: HRP-66005 ) (anti-His6 antibody replaced the discontinued GE Healthcare product listed above)
    4. Goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma-Aldrich, catalog number: A5153-1ML )
    5. Alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma-Aldrich, catalog number: A9469-2MG )
  60. Nonfat dry milk (instant, powdered) (Publix, Lakeland, FL item)
  61. Tropix CDP-Star chemiluminescent substrate (12.5 mM concentrate) (Applied BioSystems, Thermo Fisher Scientific, InvitrogenTM, catalog number: T2304 )
  62. cOmplete His-tag purification resin (Sigma-Aldrich, Roche Diagnostics, catalog number: 5893682001 )
  63. Sodium phosphate monobasic monohydrate (H2NaO4P·H2O) (Fisher Chemical, Fisher Scientific, catalog number: S369-1 )
  64. Potassium phosphate monobasic (KH2PO4) (Fisher Chemical, Fisher Scientific, catalog number: P285-500 )
  65. Ethylenediaminetetraacetic acid (EDTA) (Fisher BioReagents, Fisher Scientific, catalog number: BP120-500 )
  66. EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Roche Diagnostics, catalog number: 11873580001 )
  67. SYPRO Ruby protein gel stain (Bio-Rad Laboratories, catalog number: 1703125 )
  68. Bio-Safe Coomassie stain (Bio-Rad Laboratories, catalog number: 1610786 )
  69. Potassium sulfate (K2SO4) (Fisher Chemical, Fisher Scientific, catalog number: P304-3 )
  70. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Chemical, Fisher Scientific, catalog number: C79-500 )
  71. Bacto dehydrated culture media additive: Tryptone (BD, BactoTM, catalog number: 211705 )
  72. BBL dehydrated culture media additive: Yeast extract (BD, BBLTM, catalog number: 211929 )
  73. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  74. Agar (Sigma-Aldrich, catalog number: A7002 )
  75. Tris-base (Fisher BioReagents, Fisher Scientific, catalog number: BP152-1 )
  76. Concentrated HCl (Fisher Chemical, Fisher Scientific, catalog number: A481-212 )
  77. Sodium dodecyl sulfate (SDS) (Fisher BioReagents, Fisher Scientific, catalog number: BP166-500 )
  78. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
  79. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  80. Acrylamide/bis-acrylamide (37.5:1, 40%) (electrophoresis grade) (Fisher BioReagents, Fisher Scientific, catalog number: BP1410-1 )
  81. Tetramethylethylenediamine (TEMED, electrophoresis grade) (Fisher BioReagents, Fisher Scientific, catalog number: BP150-100 )
  82. Ammonium persulfate (APS) (Bio-Rad Laboratories, catalog number: 1610700 )
  83. 2-(N-Morpholino)ethanesulfonic acid (MES) hydrate (ACROS Organics, catalog number: 172595000 )
  84. Methanol (Fisher Chemical, Fisher Scientific, catalog number: A413-4 )
  85. Tween 20 (molecular biology grade) (Sigma-Aldrich, catalog number: P9416 )
  86. Glycine (Bio-Rad Laboratories, catalog number: 1610718 )
  87. Amersham ECL prime Western blotting detection reagent (ECL Prime) (GE Healthcare, catalog number: RPN2232 )
  88. Precision plus protein dual color standards (500 µl) (Bio-Rad Laboratories, catalog number: 1610374 )
  89. ATCC974 medium (see Recipe 1)
  90. LB medium (see Recipe 2)
  91. Lysis buffer for HisTrap HP chromatography (see Recipe 3)
  92. Lysis buffer for StrepTrap HP chromatography (see Recipe 4)
  93. Tris-salt buffer (see Recipe 5)
  94. Concentrated assay buffer (see Recipe 6)
  95. 2x SDS reducing buffer (see Recipe 7)
  96. 12% SDS-PAGE gels (see Recipe 8)
  97. 10x running buffer (see Recipe 9)
  98. Transblot buffer (see Recipe 10)
  99. 10x TBS (see Recipe 11)
  100. Tris-buffered saline with 0.1% Tween 20 (TBST) (see Recipe 12)

Equipment

  1. Sorvall Evolution RC centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Evolution RC, catalog number: 728211 ) (used to centrifuge samples at 4 to 15 °C and ≤ 12,000 x g)
  2. Fiberlite F9-4x 1000y fixed angle superspeed rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 76981 ) and Sorvall SS-34 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28020 ) (used with Sorvall Evolution RC to centrifuge 1 L cultures and 50 ml cell extract, respectively)
  3. French Press G-M (Glen Mills, model: Model 11, catalog number: 5500-000011 ) (used for lysis of cells at 20,000 to 24,000 psi)
  4. French Press standard pressure cell (35 ml, 40,000 psi) (Glen Mills, catalog number: 6800-FA-032 ) (used with French Press, item 3)
  5. Sorvall RC-3 general purpose centrifuge (Newton, CT) (used for centrifugal filtration with an HL-8 swinging bucket rotor at 4 °C and 4,000 x g)
  6. Water bath (LAUDA-Brinkmann, model: LAUDA Aqualine AL 12 , catalog number: L000610) (used to incubate reactions at 45 °C)
  7. Two bench-top microcentrifuges (Eppendorf, model number: 5418 , catalog number: 022620304) (used for centrifugation of 1-2 ml samples at up to 10,000 x g) (place one centrifuge in the cold room for 4 °C and the other on the bench-top for room temperature)
  8. BenchRocker 2D rocker (Alkali Scientific, catalog number: RS7235 )
  9. Autoclave (Consolidated Sterilizer Systems, model: SR-24C )
  10. Mini-PROTEAN Tetra Handcast Systems (Bio-Rad Laboratories, catalog number: 1658005 )
  11. SmartSpec 3000 Plus spectrophotometer (Bio-Rad Laboratories, catalog number: 1702525 ) (use for BCA assay at A562 nm and monitoring of growth at OD600)
  12. PowerPac Basic 300 V power supply (Bio-Rad Laboratories, catalog number: 1645050 ) (used for transblot overnight at 20 V and SDS-PAGE for up to 90 min at 150 V)
  13. New Brunswick I24 shaker, 3/4" orbit (7-60 °C) (Eppendorf, New BrunswickTM, model: I24 , catalog number: M1344-0000) (used for culturing cells at 37 to 42 °C at 200 rpm)
  14. Refrigerator (4 °C) (Frigidare, model: FRU17B2JW )
  15. Puffer Hubbard (-20 °C) (Revco Tech, model: 1UF1821A14 )
  16. Ultra-low temperature freezer (-80 °C) (Eppendorf, New BrunswickTM, model: C660-86 )
  17. XP-Series toploading balance (Denver Instrument, model: XP-600 )
  18. Analytical balance (Mettler Toledo, model: B balance line, AB54 )
  19. Vortex mixer (Thermolyne, catalog number: 37600 )
  20. Chemical fume hood (St. Charles Manufacturing, St. Charles, IL)
  21. Pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin type LTS)
  22. pH meter (Corning, model: Model 320) (used to titrate buffers to pH 6.8 to 8.8)
  23. Scanner (Epson Perfection, model: 3170 Photo ) (used for scanning exposed X-ray film and Coomassie Blue R250 stained protein gels at 300-600 dpi)
  24. Mini trans-blot module (Bio-Rad Laboratories, catalog number: 1703935EDU )
  25. Konica X-ray film processor (Konishiroku Photo Industry, model: QX60A )
  26. Electrophoresis systems autoradiography cassette (Fisher Scientific, catalog number: FBXC-810 )
  27. Siemens Vantage Reverse Osmosis Systems (Siemens, model: M21 series, model number: M21R004EA ) with EVOQUA filters (Siemens, catalog number: C1207098 ) and Atlantic Ultraviolet Germicidal UV Equipment (Atlantic Ultraviolet, model: MP49 ) (use water purification system to generate deionized water)
  28. BioLogic DuoFlow 10 System (Bio-Rad Laboratories, catalog number: 7600037 ) (used for protein chromatography by step gradient at flow rates of 0.5 to 1.2 ml∙min-1 and monitoring of protein fractions by A280)
  29. Stirring hotplate (PC-220 Pyroceram) (Corning, catalog number: 6795-220 ) used with magnetic stir bars (Octagonal Magnetic Stir Bar Kit) (Fisherbrand, Fisher Scientific, catalog number: 14-513-82 )

Procedure

  1. Purify proteins for in vitro reconstitution assay
    1. Prepare the H. volcanii cells for MsrA-StrepII and His6-UbaA purification as follows.
      1. Using a sterile toothpick, streak H. volcanii LR03-pJAM3010 (MsrA-StrepII) and LR03-pJAM1209 (His6-UbaA) from -80 °C glycerol stocks onto agar plates of ATCC974 medium with novobiocin (0.2 μg∙ml-1) (ATCC+Nv medium, Recipe 1). The stocks consist of stationary phase cells frozen in ATCC+Nv medium with 15 % (v/v) glycerol.
      2. Incubate the plates in plastic zip-lock bags at 42 °C for 5 days until isolated colonies appear.
      3. Using a sterile loop, transfer the isolated colonies from the plates into 4 x 25 ml ATCC+Nv medium (in 250 ml baffled culture flasks covered in foil). Grow the cells at 42 °C with rotary shaking at 200 rpm to log-phase (OD600 of 0.6-0.8).
      4. Transfer the log phase Hfx. volcanii cells to fresh 4 x 1-L ATCC+Nv medium (in 2.8-L Fernbach flasks covered in foil). Grow the cells to stationary phase (OD600 of 3.5-4.0) at 42 °C with rotary shaking at 200 rpm.
        Note: Ectopic expression of MsrA-StrepII and His6-UbaA are constitutive in the H. volcanii strains; hence, an inducer is not required.
    2. Prepare the E. coli cells for MsrA-StrepII and Flag-His6-SAMP2 purification as follows.
      1. Freshly transform plasmids pJAM3200 (for purification of MsrA-StrepII) and pJAM1132 (for purification of Flag-His6-SAMP2) into E. coli Rosetta (DE3) by chemical transformation as previously reported (Hanahan et al., 1991).
      2. Select for transformants by spread plating cells onto solid LB medium (Recipe 2) with kanamycin (50 μg∙ml-1) (LB+Km medium) and incubating the plates at 37 °C for 14 h.
      3. Transfer ~100 colonies of the freshly transformed cells into 25 ml LB+Km liquid medium (in 250 ml baffled culture flasks covered in foil) by adding 5 ml LB+Km medium to the plate using a 10 ml pipettor for resuspension and transfer.
        Note: Not all proteins require this type of approach but we do find that a subset of the proteins we work with are not readily expressed in E. coli if the cells are not freshly transformed. Note that some proteins are expressed in E. coli using the pET system even in the absence of IPTG which can cause mild toxicity and selection for cells that display loss of target gene expression. We did not want to take this type of risk that the MsrA and/or other proteins of this study would be of low level expression in E. coli.
      4. Grow the cells in the 25 ml medium at 37 °C with rotary shaking at 220 rpm to log phase (OD600 of 0.6-0.8).
      5. Subculture the cells into 500 ml fresh LB+Km medium (in 2.8 L Fernbach flasks covered in foil) to a final OD600 0.01 and grow the cells to log phase (OD600 of 0.6-0.8) at 37 °C with rotary shaking at 220 rpm.
      6. Induce protein expression in E. coli by addition of 0.4 mM IPTG. After induction, shift cell cultures to 25 °C for 12 h at 200 rpm prior to harvest.
    3. Harvest the H. volcanii and E. coli cells by centrifugation (4,500 x g, 10 min, 15 °C) using a Sorvall Evolution RC centrifuge with a Fiberlite F9-4x 1000y fixed angle superspeed rotor.
    4. Resuspend the cells at 4-5 vol per wet weight of cell pellet in lysis buffer formulated for HisTrap or StrepTrap HP chromatography (Recipes 3 and 4).
    5. Passage the resuspended cell material 4-6 times through a chilled French Press cell with a French Press at 24,000 psi. Collect the cell lysate in 50 ml polypropylene Nalgene centrifuge tubes on ice.
    6. Clarify the cell lysate by centrifugation (12,000 x g, 20 min, 4 °C) using a Sorvall Evolution RC centrifuge with a Sorvall SS-34 fixed angle rotor.
    7. Filter the cell lysate using SFCA syringe filters (0.8 μm then 0.45 µm pore size).
    8. Perform all chromatography steps at room temperature (as halophilic proteins are typically thermotolerant). Equilibrate the columns with 5-10 column volumes of Tris-salt buffer pH 7.4 (Recipe 5).
    9. Warm the clarified lysate to room temperature and apply the sample to a HisTrap HP column (5 ml bed volume, GE Healthcare) at a flow rate of 1 ml∙min-1 or StrepTrap HP column (1 ml bed volume, GE Healthcare) at a flow rate of 0.5 ml∙min-1. Control the flow rate of the chromatography using a BioLogic DuoFlow 10 System. Monitor protein fractions by A280.
    10. Remove the unbound proteins by washing the column with 140 ml Tris-salt buffer pH 7.4 at a flow rate of 1.2 ml∙min-1.
    11. Elute the bound proteins in Tris-salt buffer pH 7.4 supplemented with 5 mM d-desthiobiotin (for the StrepII tagged proteins) or 500 mM imidazole (for the His6-tagged proteins). Collect 1 ml fractions in 13 mm borosilicate glass tubes.
    12. Reducing 12% SDS-PAGE can be carried out using small portions of the eluted fractions (typically 5 μl is sufficient), followed by Coomassie Blue staining, to confirm the presence of the target protein in the eluted fractions.
    13. Concentrate the eluted protein fractions by centrifugal filtration using an Ultra-4 centrifugal filter unit with Ultracel-3 membrane (Merck) in an HL-8 swinging bucket rotor at 4 °C and 4,000 x g. We recommend concentrating the eluted fractions to a final volume of approximately 1 ml.
    14. Further purify the proteins by gel filtration chromatography by applying 0.5 ml of protein sample onto a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in Tris-salt buffer pH 7.4 at a flow rate of 0.3 ml∙min-1. Collect peak fractions based on A280. Repeat this chromatographic step as needed. His6-UbaA purifies as a dimer, while Flag-His6-SAMP2 and MsrA-StrepII purify as monomers.
    15. Check the purity of eluted fractions as indicated in Step A12 above. The volume of each fraction applied to the gel should be adjusted based on the A280: typically for an A280 of 0.5, 7.5 μl of protein is sufficient.
    16. Repeat Steps A13- A15 as necessary until desired purity is achieved. Usually, two successive gel filtration chromatography steps will result in a highly purified preparation of protein.
    17. Once the protein preparation is of sufficient purity, concentrate the protein using an Amicon Ultra-4 to a final volume of 100 μl or less.
    18. Measure the protein concentration by BCA Protein Assay according to the supplier using bovine serum albumin (BSA) as the protein standard. Store the purified proteins at 4 °C for future assay.
      Note: The purified proteins can be stored at 4 °C for 1 month.

  2. Preparation of cell lysate for in vitro reconstitution assay
    1. Grow cultures of Hfx. volcanii LR03 (Δsamp1 Δsamp2 Δsamp3 ΔmsrA ΔubaA) in 25 ml of ATCC974 medium similarly to Steps A1a to A1c.
    2. Subculture cells to an OD600 of 0.01 in 500 ml ATCC974 medium with and without 25 mM DMSO (in 2.8 L Fernbach flasks). Grow cells to stationary phase (OD600 of 3.5-4.0) at 42 °C with rotary shaking at 200 rpm.
    3. Harvest cells by centrifugation (4,500 x g, 10 min at 15 °C) using a Sorvall Evolution RC centrifuge with a Fiberlite F8-6 x 1000y fixed angle superspeed rotor.
    4. Resuspend cells in 8 ml of Tris-salt buffer, pH 7.4, 10 mM MgCl2, 100 µM bortezomib (LC Laboratories), and 30 μg∙ml-1 DNase I from bovine pancreas (Sigma-Aldrich). Dilute the bortezomib from a 200 mM stock of bortezomib dissolved in 99.8% (wt/vol) DMF.
    5. Lyse cells by French Press (20,000 psi) (similarly to Step A5).
    6. Clarify the cell lysate by centrifugation (12,000 x g, 20 min, 4 °C) using a Sorvall Evolution RC centrifuge with a Sorvall SS-34 fixed angle rotor.
    7. Filter the lysate by using a 0.2 μm SFCA syringe filter.
    8. Study the effect of different chemical agents on the stimulation of SAMP conjugate formation by dialyzing (3 times, at 4 °C) the cell lysate against 50 mM Tris-Cl, pH 7.4 supplemented with 2 M NaCl. As an example, load 7 ml of cell lysate into the dialysis tubing (3.5 kDa MWCO, SnakeSkin pre-wet according to the manufacturer’s instructions, Fisher Scientific). Place the sample in 4 L of buffer and stir the buffer for 1.5 to 2 h at 4 °C. Replace the dialysis buffer with fresh buffer and dialyze the sample for an additional 1.5 to 2 h at 4 °C. Repeat this last step for a total of three buffer exchanges against the sample.
    9. Measure protein concentration of cell lysate by BCA Protein Assay (Thermo Scientific, Rockville, IL) according to the supplier using bovine serum albumin (BSA, Thermo Scientific) as the protein standard. Store cell lysate at 4 °C for future assay.
      Note: We recommend using cell lysate for in vitro assay within 8 h after preparation.

  3. In vitro reconstitution of MsrA-dependent sampylation
    1. In a microcentrifuge tube, combine each component listed in the table below in the order shown.

      Notes:
      1. If the purpose is to purify and identify SAMP conjugates from the in vitro reconstitution assay, set the total reaction volume to 1.6 ml and use a 2.0 ml microcentrifuge tube.
      2. We always use freshly prepared concentrated assay buffer for every in vitro reconstitution assay. Mg-ATP, DMSO and protein components are selectively removed from the assay as controls. MsrA-StrepII can be purified from E. coli or H. volcanii LR03 (ΔmsrA ΔubaA Δsamp1/2/3, a sampylation deficient strain) with no detectable effect on conjugate formation.
      3. *Indicated molar concentration of His6-UbaA reflects the molecular mass of a His6-UbaA monomer, although His6-UbaA is purified as a dimer. For example, 5 µM* His6-UbaA is equivalent to 154 μg·ml-1 His6-UbaA.
    2. Incubate the reaction in a 45 °C water bath for 0, 2, 4, 8 and 18 h.
    3. Divide the 1.6 ml reaction sample into 2 parts (80 µl for Western blot and remaining 1.52 ml for purification of SAMP conjugates as described in Procedure D). After assay, remove salt from the 80 µl reaction mixtures using Zeba Spin Desalting Columns (7K MWCO) according to the supplier’s instructions (Thermo Scientific). Prior to application of the sample, equilibrate the column in 50 mM Tris-Cl buffer at pH 6.8. Centrifuge the column at 1,500 x g for 1 min using the Eppendorf microcentrifuge at room temperature. The final sample volume obtained is 100 µl.
    4. Terminate the reaction by mixing the desalted samples with an equal volume of 2x SDS reducing buffer (Recipe 7). Boil the samples for 3 x 5 min. Vortex for 30 sec after each boiling.
    5. Apply the reaction products to lanes 2-10 and the precision plus protein dual color molecular mass standards (2.5 μl) to lane 1 of reducing 12% SDS-PAGE gels (Recipe 8). The volume of sample applied may need to be adjusted based on detection sensitivity; good starting points are 3.5 µl for detection by anti-Flag antibody, and 6 µl for anti-StrepII or anti-His antibody. Separate the samples by electrophoresis at room temperature in 1x running buffer (diluted from 10x stock, Recipe 9) at 150 V for 70-90 min.
    6. Transfer the proteins to PVDF membrane at 4 °C using the mini trans-blot module in transblot buffer (Recipe 10) according to the supplier’s instructions (Bio-Rad) for 3 h at 90 V or overnight at 20 V.
    7. Remove the membrane from the cassette. Mark the location of the gel and the protein standards on the membrane using a pencil.
    8. Place the membrane upright in an 18 by 10 cm plastic container. Briefly rinse the membrane with 25 ml of 1x TBS.
    9. Block the membrane for 1 h at room temperature or overnight at 4 °C in 25 ml blocking buffer composed of TBST buffer (Recipe 11-12) supplemented with 10% (w/v) nonfat dry milk (Publix). Slowly rock the membrane during this blocking step on the medium setting of the BenchRocker 2D Rocker (Alkali Scientific Inc).
    10. Add the primary antibody to 10 ml of the blocking buffer in the following dilutions: 1:5,000 of the anti-Strep-tag II antibody to detect MsrA-StrepII, 1:5,000 of the anti-His IgG2 monoclonal antibody to detect His6-UbaA, and 1:10,000 of the alkaline phosphatase-linked anti-Flag M2 monoclonal antibody to detect Flag-His6-SAMP2. Incubate the membrane with the primary antibody solution for 60 min at room temperature using the medium setting of the rocker.
    11. Rinse the membrane for 3 x 15-30 min at room temperature with 25-50 ml TBST buffer using the high setting of the rocker.
    12. To detect the anti-Strep-tag II and anti-His primary antibodies, incubate the membrane with a 1:10,000 dilution of the secondary antibody [goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody] in blocking buffer and rinse the membrane 3 x in TBST buffer as described above.
    13. Visualize the antibody: protein complexes on the PVDF membrane by chemiluminescence using CDP-Star (Thermo Fisher Scientific) and exposure to X-ray film (Thermo Fisher Scientific) according to the supplier’s guidelines.
      Note: The HRP-conjugated His6 monoclonal antibody can be used in place of the discontinued anti-His IgG2 monoclonal antibody. HRP activity is detected using ECL Prime with X-ray film according to the user guidelines.

  4. Purification of SAMP-conjugates from the in vitro reconstitution assay
    1. Purify sampylated proteins from the in vitro reconstitution assay by use of Flag-His6-SAMP2 as the substrate and cOmplete His-tag resin (Roche) as the purification resin.
    2. Equilibrate 200 μl of the His-tag resin (Roche) in phosphate-buffered saline (PBS) (50 mM NaH2PO4, 300 mM NaCl, pH 8.0).
    3. Mix the sample (1.5 ml) after the in vitro assay with 8.5 ml PBS [5 mM EDTA, 5 mM DTT, 1 mg∙ml-1 EDTA-free protease inhibitor cocktail (Roche), 50 mM NaH2PO4, 300 mM NaCl, pH 8.0] prior to application to the His-tag resin (200 µl). To enhance the protein yield, apply the flow-through of the sample to resin for one more time.
      Note: Total reaction volume is 1.6 ml. Use 0.1 ml to analyze SAMP conjugation reaction products by Western blot and the remaining 1.5 ml to purify SAMP conjugates.
    4. Remove nonspecific proteins by washing the His-tag resin with 40 column volumes of PBS buffer.
    5. Elute bound proteins from the resin by addition of 80 μl of PBS supplemented with 200 mM imidazole.
    6. Transfer the resin with elution buffer into a new microcentrifuge tube and rotate for 20 min at 4 °C. Centrifuge the sample at 10,000 x g for 10 min using the Eppendorf microcentrifuge at 4 °C (in the cold room).
    7. Carefully remove 70 μl of the supernatant without touching the resin.
    8. Mix the supernatant with an equal volume of 2x SDS reducing buffer and boil 2 x 5 min with 30 sec vortex after each boiling.
    9. Separate proteins by 12% SDS-PAGE. Analyze the samples in parallel gels by immunoblotting and staining for total protein by SYPRO Ruby (Bio-Rad) followed by Bio-Safe Coomassie (Bio-Rad) as directed by the manufacturer. Excise the major band of SAMP-conjugates in gel slices for mass spectrometry analysis. Include gel slices of the negative control in this analysis.
      *Note: Apply 10 μl of the sample for total protein staining and subsequent excision for mass spectrometry analysis. For immunoblotting analysis, apply 3.5 µl for detection by anti-Flag antibody, and 6 µl for anti-StrepII or anti-His antibody.

Data analysis

Systematically add different components in the vitro assay to study the sampylation process. Terminate reactions at different time points to determine the optimal reaction time. In vitro reconstitution of MsrA-dependent sampylation has been published in mBio (Fu et al., 2017). Figure 1 for the representative data is originally published as Figure 4A in our previous paper (Fu et al., 2017). The 50 kDa band corresponds to monosampylated UbaA (Figure 1, lane 12). MsrA-dependent SAMP conjugates distinct from automodified UbaA were observed when all the components were added in the presence of DMSO (Figure 1, lane 14 and 19).


Figure 1. MsrA-dependent sampylation by in vitro reconstitution. Purified MsrA-StrepII was incubated with Flag-His6-SAMP2, His6-UbaA, and ATP (4 mM) for 0-18 h at 45 °C. DMSO was at a concentration of 25 mM. The reactions were supplemented with cell lysate of Hfx. volcanii LR03 (ΔmsrA ΔubaA Δsamp1/2/3, a sampylation deficient strain). Proteins after assay were separated by reducing 12% SDS-PAGE and analyzed by anti-StrepII, anti-Flag and anti-N-terminal His immunoblotting (IB) as indicated on the left. Migration of the molecular mass standards (Mr, kDa) is indicated on the left. Migration of SAMP2, SAMP2 conjugates, MsrA, UbaA, and sampylated UbaA (◄ UbaA*, the latter modified independent of MsrA) is noted on the right. The dataset of this figure was originally published in Fu et al. (2017).

Notes

Please note that our current understanding of sampylation in Archaea is incomplete. Thus, we use cell lysate of Hfx. volcanii LR03 (ΔmsrA ΔubaA Δsamp1/2/3, a sampylation deficient strain) as the resource for protein substrates and additional missing components required for sampylation. Future studies will be important to identify all the components required for sampylation and conduct complete in vitro reconstitution of sampylation using only the purified components of the system.

Recipes

  1. ATCC974 medium
    1. Dissolve 125 g NaCl, 50 g MgCl2·6H2O, 5 g K2SO4, 0.132 g CaCl2·2H2O, 5 g tryptone, and 5 g yeast extract in 750 ml deionized H2O. The medium will appear as a clear solution when all the chemicals are fully dissolved
    2. Adjust the pH of the solution to 6.8 by adding 0.5 N NaOH drop by drop
    3. Adjust the volume of the solution to final 1 L
    4. Autoclave on liquid cycle for 25 min 
  2. LB medium
    1. Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L deionized water
    2. Autoclave on liquid cycle for 25 min

Note: For Recipes 1 and 2, add antibiotics (as a powder) at the appropriate concentration to cooled media. For plates, add 15 g agar per L of media prior to autoclaving. After autoclaving, cool the media to 60 °C for 1-2 h and then dispense ~20 ml of the media per culture plate. Let the plates dry with lids on for 1-2 days at room temperature. Store liquid media in sterile bottles at room temperature or 4 °C in the dark (the latter if the media contains antibiotics or is a solid plate). Store plates and liquid cultures of H. volcanii at room temperature (not at 4 °C, as halophilic Archaea are sensitive to cold shock). Warm all media to room temperature prior to use.

  1. Lysis buffer for HisTrap HP affinity chromatography
    50 mM Tris-Cl, pH 7.4
    2 M NaCl
    40 mM imidazole
    1 mg∙ml-1 EDTA-free protease inhibitor cocktail (Roche)
  2. Lysis buffer for StrepTrap HP chromatography
    50 mM Tris-Cl, pH 7.4
    2 M NaCl
    1 mg∙ml-1 EDTA-free protease inhibitor cocktail (Roche)
  3. Tris-salt buffer
    50 mM Tris base
    2 M NaCl
    Titrated to pH 7.4 or 7.5 with concentrated HCl as indicated
  4. Concentrated assay buffer
    50 mM Tris-Cl, pH 7.5
    40 mM ATP
    250 mM DMSO
    20 mM MgCl2
    5 mM DTT
    2 M NaCl
  5. 2x SDS reducing buffer
    100 mM Tris-Cl buffer at pH 6.8
    4% (w/v) SDS
    20% (v/v) glycerol
    0.6 mg·ml-1 bromophenol blue
    5% (v/v) β-mercaptoethanol
  6. 12% SDS-PAGE gel
    1. Cast gel in Mini-PROTEAN Tetra Handcast System according to Bio-Rad with the following points of clarity
    2. Separating gel: 3.4 ml deionized H2O, 2.4 ml 40% acrylamide, 2 ml 1.5 M Tris pH 8.8, 80 μl 10% SDS, 80 μl 10% APS, and 8 μl TEMED
    3. Layer the top of the stacking gel with deionized H2O prior to the first polymerization
    4. Stacking gel: 2.9 ml deionized H2O, 0.75 ml 40% acrylamide, 1.25 ml 0.5 M Tris pH 6.8, 50 μl 10% SDS, 10% APS, and 5 μl TEMED
    5. The gel can be wrapped in wet Kimwipe and plastic wrap and stored at 4 °C for up to 1 week
  7. 10x running buffer
    1. Dissolve 30 g of Tris base, 144 g of glycine, and 10 g of SDS in 1 L of dH2O
    2. No pH adjustment is required. The pH of the buffer should be 8.3
    3. Store at room temperature and dilute to 1x before use
  8. Transblot buffer
    500 ml 0.2 M MES buffer pH 6
    1 L 100% methanol
    8.5 L deionized water
    Store at 4 °C
  9. 10x TBS (concentrated Tris-buffered saline)
    1. Dissolve 24 g Tris base and 88 g NaCl in 900 ml distilled H2O
    2. Adjust the pH to 7.6 with 12 N HCl
    3. Add distilled water to a final volume of 1 L
  10. Tris-buffered saline with 0.1% Tween 20 (TBST)
    Mix 100 ml of TBS 10x with 900 ml distilled H2O and 1 ml Tween 20

Acknowledgments

This work was funded by US Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DE-FG02-05ER15650), NIH | National Institute of General Medical Sciences (NIGMS) (NIH R01 GM57498-15) and USDA National Institute of Food and Agriculture (Hatch 1005900). This protocol was adapted from previous work published in Fu et al. (2017). The authors have no conflict of interest or competing interest to declare.

References

  1. Fu, X., Adams, Z., Liu, R., Hepowit, N. L., Wu, Y., Bowmann, C. F., Moskovitz, J. and Maupin-Furlow, J. A. (2017). Methionine sulfoxide reductase A (MsrA) and its function in ubiquitin-like protein modification in Archaea. MBio 8(5).
  2. Glickman, M. H. and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2): 373-428.
  3. Hanahan, D., Jessee, J. and Bloom, F. R. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204: 63-113.
  4. Hepowit, N. L., de Vera, I. M., Cao, S., Fu, X., Wu, Y., Uthandi, S., Chavarria, N. E., Englert, M., Su, D., Sll, D., Kojetin, D. J. and Maupin-Furlow, J. A. (2016). Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea. FEBS J 283(19): 3567-3586.
  5. Komander, D. and Rape, M. (2012). The ubiquitin code. Annu Rev Biochem 81: 203-229.
  6. Maupin-Furlow, J. A. (2014). Prokaryotic ubiquitin-like protein modification. Annu Rev Microbiol 68: 155-175.
  7. Zhao, Q., Liu, L. and Xie, Q. (2012). In vitro protein ubiquitination assay. Methods Mol Biol 876: 163-172.

简介

泛素样(Ubl)蛋白广泛分布于古细菌中并参与许多细胞途径。 为了更好地理解古细菌Ub1蛋白质修饰的过程,重建体外古细菌Ubl蛋白质缀合物的完善方法是很重要的。 该协议描述了Ubl蛋白质修饰的体外重建以及在作为模型生物的嗜盐古细菌Haloferax volcanii 中对这种修饰进行分析。

【背景】泛素(Ub)与靶蛋白共价连接的过程被称为泛素化,其控制真核细胞中大量的细胞过程(Glickman和Ciechanover,2002; Komander和Rape,2012)。遍在蛋白化由一系列酶(包括Ub激活酶(E1),Ub结合酶(E2s)和Ub连接酶(E3s))催化。泛素化的体外重建是确定酶之间或E3与蛋白质底物之间特异性的有用测定法(Zhao等人,2012)。在古细菌中,Ubl蛋白SAMP采用Ub折叠,并且与E1样酶UbaA催化的蛋白靶标异肽连接[Maupin-Furlow,(2014)综述]。尽管E1同系物在古细菌中广泛存在,但基于一级序列比较,在大多数古细菌中未预测经典E2或E3酶。我们最近对Haloferax volcanii的研究表明甲硫氨酸亚砜还原酶A(MsrA)是Ubl蛋白质修饰(sampylation)与UbaA一起在体内温和的氧化条件下和< (体外)(Fu et。,2017)。在这里,我们描述了一个详细的体外协议来重构和分析MsrA依赖性的sampylation。

关键字:古生菌, 泛素(Ub), 翻译后修饰, SAMP

材料和试剂

  1. 手套(Fisherbrand,Fisher Scientific,目录号:19-130-1597C)
  2. 无菌牙签(Royal Paper Products,Inc.,产品编号:R820)
  3. Ampac 500系列SealPAK耐用袋(10.2 x 15.2 cm)(Fisher Scientific,产品目录号:01-812-25D)(使用彩色标签胶带密封蛋白质印迹膜,CDP-Star或ECL主要试剂在小袋中)
    制造商:Ampac,产品目录号:50024。
  4. 无菌环(Fisherbrand,Fisher Scientific,目录号:22-363-599)
  5. Fernbach瓶(2.8 L,宽口)(Corning,PYREX®,<目录号:4420-2XL)
  6. 挡板培养瓶(250ml)(DWK Life Sciences,Kimble,目录号:25630-250)
  7. 铝箔(Fisherbrand,Fisher Scientific,产品目录号:01-213-105)(用于包装高压灭菌的物品)
  8. 无菌聚苯乙烯一次性血清学10毫升带放大镜条纹的移液管(Fisherbrand,Fisher Scientific,目录号:13-678-11E)
  9. 聚丙烯圆底离心管,50ml(Nalgene,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3119-0050,产品编号:UX-06327-47)
  10. 用无表面活性剂醋酸纤维素(SFCA)膜(Thermo Fisher Scientific,Thermo Scientific TM,目录号:290-3320)的Nalgene快速流动无菌一次性瓶顶部0.2μm过滤器
  11. HisTrap HP柱(5毫升)(GE Healthcare,目录号:17524802)
  12. StrepTrap HP柱(1 ml)(GE Healthcare,目录号:29048653)

  13. 13×100mm 2培养管(Fisher Scientific,目录号:14-961-27)
  14. 具有平端(13×100mm)的硼硅酸盐玻璃管(Fisherbrand,Fisher Scientific,目录号:14-961-27)(用于细胞培养)
  15. 具有Ultracel-3膜(NMWL 3 kDa)的Amicon Ultra-4离心过滤装置(Merck,目录号:UFC800308)
  16. Superdex 75 10/300 GL柱(GE Healthcare,目录号:17517401)
  17. 无表面活性剂的醋酸纤维素(SFCA)膜注射过滤器(28mm膜,孔径为0.2,0.45和0.8μm,丙烯酸外壳)(Corning,目录号:431219,431220和431221)
  18. SnakeSkin透析管(3.5K MWCO,22mm I.D.)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:68035)。
  19. 1.5ml微量离心管(Fisherbrand,Fisher Scientific,目录号:02-681-320)
  20. 2.0 ml微量离心管(Fisherbrand,Fisher Scientific,目录号:02-681-321)
  21. Zeba Spin脱盐柱(7K MWCO)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:89882)
  22. Amersham Hybond P 0.45聚偏二氟乙烯(PVDF)膜(GE Healthcare,目录号:10600023)
  23. 一次性塑料比色皿(Fisherbrand,Fisher Scientific,目录号:149-551-27)
  24. X光胶片(RPI,目录号:248300)
  25. Rack LTS提示('P20'2-20μl,'P200'20-200μl,'P1000'100-1,000μl)(Mettler-Toledo,Rainin,产品目录号:17001865,17001863和17001864)
  26. 10毫升Luer-Lok注射器(BD,目录号:309695)
  27. Kimwipes精致任务雨刷,1层(KWCC,Kimberly-Clark,目录编号:34155)
  28. 彩色标签胶带,彩虹包(Fisherbrand,Fisher Scientific,目录号:15-901-10R)
  29. 四个正方形盒(8×10“)(Fisher Scientific,目录号:FBXC-810)(用于Western印迹膜暴露于X射线胶片)
  30. 玻璃储存/介质瓶(DWK Life Sciences,Kimble,产品目录号:14395-500为500毫升,14395-1000为1,000毫升)
  31. 聚丙烯格里芬低型塑料烧杯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:1201-4000,2000毫升和1201-1000,1000毫升)
  32. 聚乙烯包装薄膜(Fisherbrand,Fisher Scientific,目录号:15-610)(可存储包裹在Kimwipes中的SDS-PAGE凝胶,用去离子水润湿并在电泳前在4°C进一步用聚乙烯薄膜包裹长达2周)
  33. 聚苯乙烯比色皿(1.5ml容量)(Fisherbrand,Fisher Scientific,目录号:14-955-127)(用于可见光谱范围,340-750nm的测定)
  34. 聚丙烯塑料量筒(Nalgene,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3664-0050,3664-0250和3664-1000)
  35. 聚丙烯瓶盖(DWK Life Sciences,Kimble,产品目录号:73660-13)(与13 mm O.D.平端培养管一起使用)
  36. 带有清洁盖子的培养皿(Fisherbrand,Fisher Scientific,目录号:FB0875712)
  37. 细胞
    1. 携带质粒pJAM3010(编码msrA-strepII质粒的质粒,该质粒在P2N1质粒的控制下)的Haloferax volcanii LR03(Δsamp1Δsamp2Δsamp3ΔmsrAΔubaA), (Fu等人,2017)或pJAM1209(一种编码他的6-tubA基因的质粒)在组成型启动子(Hepowit等人,2016)的控制下)或不携带任何质粒。
      注:H. volcanii菌株和质粒可根据要求提供。
    2. 携带质粒pJAM3200(在T7启动子控制下的编码msrA-strepII的质粒)的大肠杆菌Rosetta(DE3)(Novagen,Merck,目录号:70954-3) Maupin-Furlow实验室(Fu等人,2017)或质粒pJAM1132(在T7控制下编码flag-his6-samp2的质粒)启动子)来自Maupin-Furlow实验室(Hepowit et al。,2016)
      注意:质粒可以从Maupin-Furlow实验室索取。
  38. MsrA-StrepII(根据下文概述的步骤纯化)
  39. His 6 -UbaA(按照下面概述的步骤纯化)
  40. 去离子H 2 O(详情请参阅设备编号22)
  41. 甘油(Sigma-Aldrich,目录号:G5516)
  42. 新生霉素(Sigma-Aldrich,目录号:N1628)
  43. 硫酸卡那霉素(Fisher BioReagents,Fisher Scientific,目录号:BP906-5)
  44. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Fisher BioReagents,Fisher Scientific,目录号:BP1620-1)
  45. -Desthiobiotin(Sigma-Aldrich,目录号:D1411-500MG)
  46. 咪唑,ACS试剂,≥99%滴定(Sigma-Aldrich,目录号:I2399)
  47. 考马斯亮蓝R-250(Bio-Rad Laboratories,目录号:1610436)
  48. Pierce Bicinchoninic acid(BCA)蛋白质分析(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:23225)
  49. 白蛋白,牛(BSA)(冻干粉,通过琼脂糖凝胶电泳纯度≥96%)(Sigma-Aldrich,目录号:A2153)
  50. 氯化镁六水合物(MgCl 2·6H 2 O)(Fisher Chemical,Fisher Scientific,目录号:M35-12)
  51. 硼替佐米蛋白酶体抑制剂(游离碱,纯度> 99%)(LC Laboratories,目录号:B-1408)
  52. 来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:D4263-1VL),含有≥0.27mg总蛋白的含有2000个Kunitz单位的DNase I(Sigma-Aldrich,目录号:D4527)的标准化小瓶
  53. 二甲基甲酰胺(DMF)(Fisher BioReagents,Fisher Scientific,目录号:BP1160-500)
  54. 氯化钠(NaCl)(Fisher Chemical,Fisher Scientific,目录号:S642-12)
  55. 腺苷5'-三磷酸(ATP),二钠盐水合物,98%(ACROS Organics,目录号:102800100)
  56. 二甲基亚砜(DMSO),分子生物学级(Sigma-Aldrich,目录号:D8418-50ML)(用于体外试验)
  57. DMSO,ACS认证(Fisher Chemical,Fisher Scientific,目录号:D128-1)(用于培养细胞)
  58. 二硫苏糖醇(DTT)(Fisher BioReagents,Fisher Scientific,目录号:BP172-5)
  59. 抗体
    1. 链霉亲和素II单克隆抗体(小鼠)(QIAGEN,目录号:34850)
    2. 抗His IgG2单克隆抗体(小鼠)(GE Healthcare,目录号:27471001)
    3. HRP缀合的His 6单克隆抗体(Proteintech,目录号:HRP-66005)(抗His 6抗体替换上面列出的停用的GE Healthcare产品)。
    4. 山羊抗小鼠IgG(整个分子) - 碱性磷酸酶连接的抗体(Sigma-Aldrich,目录号:A5153-1ML)
    5. 连接碱性磷酸酶的抗Flag M2单克隆抗体(Sigma-Aldrich,目录号:A9469-2MG)

  60. 脱脂奶粉(速溶,粉状)(Publix,莱克兰,佛罗里达州项目)
  61. Tropix CDP-Star化学发光底物(12.5mM浓缩物)(Applied BioSystems,Thermo Fisher Scientific,Invitrogen TM,产品目录号:T2304)
  62. 完整的His标签纯化树脂(Sigma-Aldrich,Roche Diagnostics,目录号:5893682001)
  63. 磷酸二氢钠一水合物(H 2 O·NaO·4P·H 2 O)(Fisher Chemical,Fisher Scientific,目录号:S369-1)
  64. 磷酸二氢钾(KH 2 PO 4)(Fisher Chemical,Fisher Scientific,目录号:P285-500)
  65. 乙二胺四乙酸(EDTA)(Fisher BioReagents,Fisher Scientific,目录号:BP120-500)
  66. 无EDTA蛋白酶抑制剂混合物(Sigma-Aldrich,Roche Diagnostics,目录号:11873580001)
  67. SYPRO Ruby蛋白质凝胶染色剂(Bio-Rad Laboratories,目录号:1703125)
  68. 生物安全的考马斯染色剂(Bio-Rad Laboratories,目录号:1610786)
  69. 硫酸钾(K 2 SO 4)(Fisher Chemical,Fisher Scientific,目录号:P304-3)
  70. 氯化钙二水合物(CaCl 2·2H 2 O)(Fisher Chemical,Fisher Scientific,目录号:C79-500)
  71. 细菌脱水培养基添加剂:胰蛋白胨(BD,Bacto TM,目录号:211705)
  72. BBL脱水培养基添加剂:酵母提取物(BD,BBL TM,目录号:211929)
  73. 氢氧化钠(NaOH)(Fisher Scientific,目录号:BP359-212)
  74. 琼脂(Sigma-Aldrich,目录号:A7002)
  75. Tris-碱(Fisher BioReagents,Fisher Scientific,目录号:BP152-1)
  76. 浓HCl(Fisher Chemical,Fisher Scientific,目录号:A481-212)
  77. 十二烷基硫酸钠(SDS)(Fisher BioReagents,Fisher Scientific,目录号:BP166-500)
  78. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  79. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  80. 丙烯酰胺/双丙烯酰胺(37.5:1,40%)(电泳级)(Fisher BioReagents,Fisher Scientific,目录号:BP1410-1)
  81. 四甲基乙二胺(TEMED,电泳级)(Fisher BioReagents,Fisher Scientific,目录号:BP150-100)
  82. 过硫酸铵(APS)(Bio-Rad Laboratories,目录号:1610700)
  83. 2-(N-吗啉代)乙磺酸(MES)水合物(ACROS Organics,目录号:172595000)
  84. 甲醇(Fisher Chemical,Fisher Scientific,目录号:A413-4)
  85. 吐温20(分子生物学等级)(Sigma-Aldrich,目录号:P9416)
  86. 甘氨酸(Bio-Rad Laboratories,目录号:1610718)
  87. Amersham ECL Prime Western印迹检测试剂(ECL Prime)(GE Healthcare,目录号:RPN2232)
  88. 精确加蛋白双色标准品(500μl)(Bio-Rad Laboratories,目录号:1610374)
  89. ATCC974培养基(见配方1)
  90. LB培养基(见方案2)
  91. 用于HisTrap HP色谱的裂解缓冲液(见第3部分)
  92. 用于StrepTrap HP色谱的裂解缓冲液(见配方4)
  93. Tris-盐缓冲液(见方法5)
  94. 浓缩的分析缓冲液(见第6部分)
  95. 2x SDS还原缓冲液(见第7部分)
  96. 12%SDS-PAGE凝胶(见方法8)
  97. 10倍的运行缓冲区(见第9章)
  98. Transblot缓冲区(见第10节)
  99. 10倍TBS(见11配方)
  100. 含有0.1%吐温20(TBST)的Tris缓冲盐水(参见配方12)

设备

  1. Sorvall Evolution RC离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall Evolution RC,目录号:728211)(用于在4至15℃下离心样品并且≤12,000xg)
  2. Fiberlite F9-4x 1000y固定角超速转子(Thermo Fisher Scientific,Thermo Scientific TM,目录号:76981)和Sorvall SS-34固定角转子(Thermo Fisher Scientific,Thermo Scientific TM TM-目录号:28020)(与Sorvall Evolution RC一起分别用于离心1L培养物和50ml细胞提取物)
  3. 法国新闻G-M(Glen Mills,型号:Model 11,目录号:5500-000011)(用于细胞在20,000到24,000 psi的裂解)
  4. French Press标准压力传感器(35 ml,40,000 psi)(Glen Mills,产品目录号:6800-FA-032)(与French Press,第8项一起使用)
  5. Sorvall RC-3通用型离心机(Newton,CT)(用于在4°C和4,000 x的HL-8摆动斗式转子的离心过滤)
  6. 水浴(LAUDA-Brinkmann,型号:LAUDA Aqualine AL 12,目录号:L000610)(用于在45°C孵育反应)
  7. 两台台式微型离心机(Eppendorf,型号:5418,目录编号:022620304)(用于离心1-2,000ml样品,最高10,000μgxg)(将一台离心机置于冷藏室中4°C,另一个在台式室温下)
  8. BenchRocker 2D摇杆(碱性科学,目录号:RS7235)
  9. 高压灭菌器(统一灭菌器系统,型号:SR-24C)
  10. Mini-PROTEAN Tetra Handcast System(Bio-Rad Laboratories,目录号:1658005)
  11. SmartSpec 3000 Plus分光光度计(Bio-Rad Laboratories,目录号:1702525)(用于562nm处的BCA测定和监测OD 600的生长)。
  12. PowerPac Basic 300 V电源(Bio-Rad Laboratories,产品目录号:1645050)(用于在20 V下进行transblot过夜并在150 V下进行SDS-PAGE达90分钟)
  13. 新不伦瑞克I24摇床,3/4“轨道(7-60℃)(Eppendorf,New Brunswick TM,型号:I24,目录号:M1344-0000)(用于在37至42°C,200 rpm)
  14. 冰箱(4°C)(Frigidare,型号:FRU17B2JW)
  15. Puffer Hubbard(-20°C)(Revco Tech,型号:1UF1821A14)
  16. 超低温冰箱(-80℃)(Eppendorf,New Brunswick TM,型号:C660-86)
  17. XP系列顶部平衡(丹佛仪器,型号:XP-600)
  18. 分析天平(Mettler Toledo,型号:B余额线,AB54)
  19. 涡旋混合器(Thermolyne,目录号:37600)
  20. 化学通风橱(圣查尔斯制造,伊利诺伊州圣查尔斯)
  21. 移液器(2-20μl,20-200μl,100-1,000μl)(Rainin LTS)
  22. pH计(Corning,型号:320型)(用于滴定缓冲液至pH6.8至8.8)
  23. 扫描仪(Epson Perfection,型号:3170照片)(用于扫描曝光的X光胶片和考马斯蓝R250染色蛋白凝胶,分辨率300-600dpi)
  24. 迷你转印模块(Bio-Rad Laboratories,目录号:1703935EDU)
  25. 柯尼卡X光胶片处理器(Konishiroku Photo Industry,型号:QX60A)
  26. 电泳系统放射自显影盒(Fisher Scientific,目录号:FBXC-810)
  27. 西门子Vantage反渗透系统(西门子,型号:M21系列,型号:M21R004EA)带有EVOQUA过滤器(西门子,目录号:C1207098)和大西洋紫外线杀菌紫外设备(大西洋紫外线,型号:MP49)(使用水净化系统产生去离子水)
  28. BioLogic DuoFlow 10系统(Bio-Rad Laboratories,产品目录号:7600037)(用于0.5至1.2 ml·min -1的梯度梯度的蛋白质层析和通过A < sub> 280 )
  29. 使用磁力搅拌棒(八角磁性搅拌棒试剂盒)(Fisherbrand,Fisher Scientific,目录号:14-513-82)使用的搅拌热板(PC-220 Pyroceram)(Corning,目录号:6795-220)

程序

  1. 纯化蛋白进行体外重构测定
    1. 准备 H。用于如下的MsrA-StrepII和His6-UbaA纯化。
      1. 使用无菌牙签,条纹。将来自-80℃甘油储备液的火山灰-RL3-pJAM3010(MsrA-StrepII)和LR03-pJAM1209(His6-UbaA)置于含有新生霉素(ATCC974)的琼脂板上(0.2μg·ml -1)(ATCC + Nv培养基,方案1)。库存由在具有15%(v / v)甘油的ATCC + Nv培养基中冷冻的固定相细胞组成。
      2. 在42°C的温度下将塑料拉链锁袋中的培养板培养5天,直到出现分离的菌落。
      3. 使用无菌环,将分离的菌落从平板转移至4×25ml ATCC + Nv培养基(在250ml带有箔的覆盖有挡板的培养瓶中)。将细胞在42℃下以200rpm的转速振荡生长至对数生长期(OD <600> 0.6-0.8)。
      4. 转移日志阶段 Hfx。 volcanii 细胞培养至新鲜的4×1-L ATCC + Nv培养基(在覆盖箔的2.8-Fernbach瓶中)。在42℃下以200rpm旋转振荡使细胞生长至静止期(OD460-3.5-4.0)。
        注:MsrA-StrepII和His6-UbaA的异位表达在火山口菌株中是组成型的;因此,不需要诱导器。
    2. 准备 E。用于MsrA-StrepII和Flag-His6-SAMP2纯化的大肠杆菌细胞如下。
      1. 将质粒pJAM3200(用于MsrA-StrepII的纯化)和pJAM1132(用于纯化Flag-His6-SAMP2)新鲜转化为E.通过如先前报道的化学转化(Hanahan等人,1991),通过大肠杆菌Rosetta(DE3)。
      2. 用卡那霉素(50μg·ml-1)(LB + Km培养基)在固体LB培养基(配方2)上涂铺细胞,选择转化体并在37℃孵育14小时。
      3. 将约100个新鲜转化细胞的菌落转移到25ml LB + Km液体培养基中(在250ml带盖箔片的挡板培养瓶中),使用10ml移液器将5ml LB + Km培养基加入到平板中以重新悬浮并转移。
        注意:并非所有的蛋白质都需要这种类型的方法,但我们确实发现如果细胞未被新鲜转化,我们使用的蛋白质子集不易在大肠杆菌中表达。请注意,即使在不存在IPTG的情况下,使用pET系统也可在大肠杆菌中表达一些蛋白质,这可导致轻微的毒性并选择显示靶基因表达缺失的细胞。我们不希望发生这种类型的风险,即本研究的MsrA和/或其他蛋白在大肠杆菌中的表达水平低。
      4. 在37℃下在25ml培养基中培养细胞,以220rpm的旋转振荡至对数期(OD 600在0.6-0.8)。
      5. 将细胞继代培养到500ml新鲜的LB + Km培养基中(在箔中覆盖的2.8L Fernbach瓶中)至最终OD 600≤0.01,并使细胞生长至对数生长期(OD 600) > 0.6-0.8)在37℃下以220rpm旋转摇动。
      6. 在E中诱导蛋白质表达。加入0.4mM IPTG。诱导后,在收获前将细胞培养物以200rpm的速度转移至25℃12小时。
    3. 收获 H。 volcanii 和 E。使用带有Fiberlite F9-4x 1000y固定角度超速转子的Sorvall Evolution RC离心机通过离心(4,500xg,10分钟,15℃)培育大肠杆菌细胞。
    4. 在配制用于HisTrap或StrepTrap HP层析的裂解缓冲液(配方3和4)中以每湿重细胞团粒4-5体积重悬细胞。
    5. 使用French Press在24,000 psi的压力下将重新悬浮的细胞材料通过冷冻法式压力机通过4-6次。将细胞裂解液收集在冰上的50ml聚丙烯Nalgene离心管中。
    6. 使用带有Sorvall SS-34固定角转子的Sorvall Evolution RC离心机通过离心(12,000xg,20分钟,4℃)澄清细胞裂解物。
    7. 使用SFCA注射器过滤器(0.8μm,然后0.45μm孔径)过滤细胞裂解物。
    8. 在室温下进行所有色谱步骤(因为嗜盐蛋白通常是耐热的)。用5-10个柱体积的Tris-盐缓冲液pH7.4(方案5)平衡柱子。
    9. 将澄清的裂解物温热至室温,并将样品以1ml∙min -1 -1或StrepTrap HP柱(1ml)的流速应用于HisTrap HP柱(5ml床体积,GE Healthcare)床体积,通用电气医疗集团),流速为0.5 ml·min -1 -1。使用BioLogic DuoFlow 10系统控制色谱流速。通过A 280监测蛋白质组分。
    10. 通过用140ml Tris-盐缓冲液pH7.4以1.2ml·min -1 -1的流速洗涤柱来除去未结合的蛋白质。
    11. 在补充有5mM二硫键生物素(对于StrepII标记的蛋白质)或500mM咪唑(对于His6标记的蛋白质)的Tris-盐缓冲液pH7.4中洗脱结合的蛋白质蛋白质)。
      在13毫米硼硅酸盐玻璃管中收集1毫升级分
    12. 使用小部分洗脱级分(通常5μl就足够了),然后进行考马斯蓝染色,以确认洗脱级分中存在目标蛋白质,可以进行12%SDS-PAGE的降低。
    13. 使用具有Ultracel-3膜(Merck)的Ultra-4离心过滤器单元在HL-8摆动斗式转子中在4℃和4,000gxg下通过离心过滤浓缩洗脱的蛋白质级分。我们建议将洗脱的组分浓缩至最终体积约1 ml。
    14. 通过凝胶过滤色谱法进一步纯化蛋白质,将0.5ml蛋白质样品加入在Tris-盐缓冲液(pH7.4)中平衡的Superdex 75 10/300 GL柱(GE Healthcare),流速为0.3ml·min -1 。基于A 280收集峰分数。根据需要重复该色谱步骤。 His6-UbaA纯化为二聚体,而Flag-His6-SAMP2和MsrA-StrepII纯化为单体。
    15. 检查洗脱级分的纯度,如上面步骤A12所示。施加到凝胶上的每个级分的体积应基于A 280进行调节:通常对于0.5的A 280,7.5μl的蛋白质就足够了。
    16. 根据需要重复步骤A13-A15,直至达到所需的纯度。通常,两次连续的凝胶过滤色谱步骤将产生高度纯化的蛋白质制剂。
    17. 一旦蛋白质制备纯度足够高,使用Amicon Ultra-4浓缩蛋白至100μl或更少的终体积。
    18. 根据供应商使用牛血清白蛋白(BSA)作为蛋白质标准物,通过BCA蛋白质测定法测量蛋白质浓度。将纯化的蛋白质储存在4°C以备将来检测。
      注意:纯化的蛋白质可以在4°C下保存1个月。

  2. 制备用于体外重构测定的细胞裂解物
    1. 种植Hfx的文化。与步骤A1a至A1c相似,在25ml的ATCC974培养基中加入火山vol vol LR LR LR LR LR LR <<<<<<<<
    2. 在含有或不含25mM DMSO的500ml ATCC974培养基(在2.8L Fernbach瓶中)中将细胞传代至0.01的OD 600。在42℃下以200rpm旋转振荡使细胞生长至静止期(OD460-3.5-4.0)。
    3. 使用带有Fiberlite F8-6 x 1000y固定角度超速转子的Sorvall Evolution RC离心机通过离心收获细胞(4,500xg,10分钟,15℃)。
    4. 将细胞重悬于8ml Tris-盐缓冲液(pH 7.4),10mM MgCl 2,100μM硼替佐米(LC Laboratories)和30μg·ml -1 DNase I来自牛胰腺(Sigma-Aldrich)。稀释溶解于99.8%(wt / vol)DMF中的200mM硼替佐米储备液中的硼替佐米。
    5. 通过French Press(20,000psi)的溶胞细胞(类似于步骤A5)。
    6. 使用具有Sorvall SS-34固定角转子的Sorvall Evolution RC离心机通过离心(12,000xg,20分钟,4℃)澄清细胞裂解物。
    7. 使用0.2μmSFCA注射器过滤器过滤裂解物。
    8. 通过透析(3次,在4℃)细胞裂解物对补充有2M NaCl的50mM Tris-Cl,pH 7.4,研究不同化学试剂对刺激SAMP缀合物形成的作用。例如,将7ml细胞裂解液装入透析管(3.5kDa MWCO,根据制造商说明书Fisher Scientific预湿的SnakeSkin)中。将样品放入4L缓冲液中,并在4℃下搅拌缓冲液1.5至2小时。用新鲜缓冲液替换透析缓冲液,并在4°C下透析样品1.5至2小时。对样品重复最后一步,共进行三次缓冲液交换。
    9. 根据供应商使用牛血清白蛋白(BSA,Thermo Scientific)作为蛋白质标准物,通过BCA蛋白质测定法(Thermo Scientific,Rockville,IL)测量细胞裂解物的蛋白质浓度。将细胞裂解液储存在4°C以备将来检测。
      注意:我们建议在制备后8小时内使用细胞裂解液进行体外测定。

  3. MsrA依赖性sampylation体外重建
    1. 在微量离心管中,按照显示的顺序将下表中列出的各组分合并。

      备注:
      1. 如果目的是从体外重建测定中纯化和鉴定SAMP偶联物,将总反应体积设定为1.6ml并使用2.0ml微量离心管。
      2. 我们总是使用新鲜制备的浓缩测定缓冲液用于每个体外重建测定。选择性地从测定中除去作为对照的Mg-ATP,DMSO和蛋白质组分。 MsrA-StrepII可从E.coli或H. volcanii LR03(ΔmsrAΔubaAΔsamp1/ 2/3,一种sampylation缺陷菌株)中纯化,对偶联物形成没有可检测的影响。
      3. * His 6 -UbaA的指示摩尔浓度反映了His 6 -UbaA单体的分子量,尽管His 6 -UbaA被纯化为二聚体。例如,5μM* His6-UbaA相当于154μg·ml-1 His6-UbaA。
    2. 将反应液在45°C水浴中孵育0,2,4,8和18小时。
    3. 将1.6ml反应样品分成2份(80μl用于蛋白质印迹,剩余1.52ml用于如步骤D所述纯化SAMP缀合物)。分析后,根据供应商的说明书(Thermo Scientific),使用Zeba Spin Desalting Columns(7K MWCO)从80μl反应混合物中除去盐。在应用样品前,在pH 6.8的50 mM Tris-Cl缓冲液中平衡柱子。使用Eppendorf微量离心机在室温下将柱子在1,500xg 离心1分钟。
      最终样品体积为100μl
    4. 通过将脱盐的样品与等体积的2x SDS还原缓冲液混合来终止反应(配方7)。煮沸样品3 x 5分钟。每次沸腾后涡旋30秒。
    5. 将反应产物应用于泳道2-10,并将精确加蛋白双色分子量标准品(2.5μl)应用于还原12%SDS-PAGE凝胶的泳道1(配方8)。施加的样品量可能需要根据检测灵敏度进行调整;良好的起点是3.5μl用于通过抗Flag抗体进行检测,并且6μl用于抗StrepII或抗His抗体。通过在室温下在1×运行缓冲液(从10×储备液,配方9稀释)中在150V电泳分离样品70-90分钟。
    6. 根据供应商的说明书(Bio-Rad),在90 V下使用迷你trans-blot模块在transblot缓冲液(配方10)中将蛋白转移至PVDF膜上4°C,或在20 V下过夜。
    7. 从盒中取出膜。
      使用铅笔标记凝胶的位置和膜上的蛋白质标准。
    8. 将膜直立放入18 x 10厘米的塑料容器中。用25ml 1×TBS简单冲洗膜。
    9. 在室温下封闭膜1小时或在4℃在25ml封闭缓冲液中过夜,所述封闭缓冲液由补充有10%(w / v)脱脂奶粉(Publix)的TBST缓冲液(配方11-12)组成。在BenchRocker 2D Rocker(Alkali Scientific Inc)的中型设置中,在阻挡步骤中缓慢摇动膜片。
    10. 按以下稀释度将一抗加入到10ml封闭缓冲液中:1:5,000的抗Strep-tag II抗体以检测MsrA-StrepII,1:5,000的抗-His IgG2单克隆抗体以检测His-sub > 6-UbaA和1:10,000的碱性磷酸酶连接的抗Flag M2单克隆抗体以检测Flag-His 6 -SAMP2。使用摇杆的中等设置,在室温下孵育膜与主要抗体溶液60分钟。
    11. 使用摇杆的高设置,用25-50ml TBST缓冲液在室温下冲洗膜3×15-30分钟。
    12. 为了检测抗Strep标签II和抗His一抗,将膜与二抗的抗体(山羊抗小鼠IgG(全分子) - 碱性磷酸酶连接的抗体)的1:10,000稀释液在封闭缓冲液中孵育并且如上所述在TBST缓冲液中冲洗膜3次。
    13. 根据供应商指南,使用CDP-Star(赛默飞世尔科技)通过化学发光和暴露于X射线胶片(Thermo Fisher Scientific)在PVDF膜上显现抗体:蛋白质复合物。
      注意:可以使用HRP缀合的His 6单克隆抗体代替中止的抗His IgG2单克隆抗体。根据用户指南,使用带有X光胶片的ECL Prime检测HRP活性。

  4. 从体外重建试验纯化SAMP-缀合物
    1. 通过使用Flag-His 6 -SAMP2作为底物并且使用完全His标签树脂(Roche)作为纯化树脂从体外重构测定法中纯化标准蛋白质。
    2. 在磷酸盐缓冲盐水(PBS)(50mM NaH 2 PO 4,300mM NaCl,pH 8.0)中平衡200μlHis-标签树脂(Roche)。
    3. 在体外测定后,将样品(1.5ml)与8.5ml PBS [5mM EDTA,5mM DTT,1mg / ml -1无EDTA蛋白酶抑制剂鸡尾酒(Roche),50mM NaH 2 PO 4,300mM NaCl,pH 8.0]中,然后施加到His-标签树脂(200μl)上。
      为了提高蛋白质产量,将样品流过树脂一次 注意:总反应体积为1.6毫升。使用0.1ml通过蛋白质印迹分析SAMP缀合反应产物,剩余的1.5ml用于纯化SAMP缀合物。
    4. 用40倍柱体积的PBS缓冲液洗涤His-tag树脂去除非特异性蛋白质。
    5. 通过加入80μl补充有200mM咪唑的PBS从树脂中洗脱结合的蛋白质。
    6. 用洗脱缓冲液将树脂转移到一个新的微量离心管中,并在4℃下旋转20分钟。使用Eppendorf微量离心机在4℃下(在冷室中)将样品在10,000×g下离心10分钟。
    7. 小心取出70μl的上清液,不要触摸树脂。
    8. 将上清液与等体积的2x SDS还原缓冲液混合,并在每次沸腾后用30秒漩涡煮沸2 x 5分钟。
    9. 通过12%SDS-PAGE分离蛋白质。按照制造商的指示,通过免疫印迹法和通过SYPRO Ruby(Bio-Rad)随后Bio-Safe Coomassie(Bio-Rad)染色总蛋白质来分析平行凝胶中的样品。在凝胶切片中切下主要的SAMP-缀合物条带进行质谱分析。
      在此分析中包含阴性对照的凝胶片。
      注意:将10μl样品用于总蛋白染色和随后的切除以进行质谱分析。对于免疫印迹分析,应用3.5μl用于通过抗Flag抗体进行检测,并且用6μl用于抗StrepII或抗His抗体。

数据分析

系统地在体外试验中添加不同的组分以研究其过程。终止不同时间点的反应以确定最佳反应时间。 MsrA依赖性sampylation的体外重构已经在mBio中发表(Fu等人,2017)。代表性数据的图1最初发表在我们以前的论文(Fu等人,2017)中的图4A中。 50kDa的条带对应于单腺苷酸化的UbaA(图1,泳道12)。当在DMSO存在下加入所有组分时观察到与自修饰的UbaA不同的MsrA依赖性SAMP缀合物(图1,泳道14和19)。


图1.纯化的MsrA-StrepII与Flag-His孵育后的MsrA-StrepII复合物。图1.纯化的MsrA-StrepII与Flag-His 6-SAMP2,His6-UbaA和ATP(4mM)在45℃下孵育0-18小时。 DMSO浓度为25mM。反应物补充有hfx的细胞裂解物。 volcanii LR03(ΔmsrAΔubaAΔsamp1/ 2/3,一种桑白酶缺陷型菌株)。测定后的蛋白质通过减少12%SDS-PAGE进行分离,并通过左侧所示的抗StrepII,抗Flag和抗N-末端His免疫印迹(IB)进行分析。分子量标准(M r,kDa)的迁移在左侧标出。右边注明了SAMP2,SAMP2结合物,MsrA,UbaA和sampylated UbaA(◄UbaA *,后者修改为独立于MsrA)的迁移。该数据集最初发表于Fu等人(2017)。

笔记

请注意,我们目前对古细菌中的sampylation的了解并不完整。因此,我们使用hfx的细胞裂解液。 volcanii LR03(ΔmsrAΔubaAΔsamp1/ 2/3,一种桑白酶缺陷型菌株)作为蛋白质底物的资源和另外的缺失组分需要进行sampylation。未来的研究将非常重要,以确定所需的所有组分进行sampylation并进行完整的体外重建sampylation只使用系统的纯化组件。

食谱

  1. ATCC974培养基
    1. 溶解125g NaCl,50g MgCl 2·6H 2 O,5g K 2 SO 4,0.132 g CaCl 2·2H 2 O,5g胰蛋白胨和5g酵母提取物在750ml去离子H 2 O中的溶液。当所有的化学物质都完全溶解后,培养基就会变成清澈的溶液。

    2. 通过逐滴加入0.5 N NaOH将溶液的pH调节至6.8
    3. 调整溶液的体积至最终1 L
    4. 液体循环高压灭菌25分钟
  2. LB培养基

    1. 10克胰蛋白胨,5克酵母提取物和10克NaCl溶于1升去离子水中
    2. 液体循环高压灭菌25分钟
    注意:对于配方1和2,将适量浓度的抗生素(作为粉末)加入冷却的培养基中。对于平板,在高压灭菌之前,每升培养基加入15克琼脂。高压灭菌后,将培养基冷却至60°C 1-2小时,然后每培养皿分配约20毫升培养基。让板在室温下盖上盖子干燥1-2天。将液体培养基在室温或4°C黑暗条件下(无论培养基中含有抗生素还是固体培养皿)储存在无菌瓶中。由于嗜盐古细菌对冷休克敏感,所以在室温下在4℃下储存板状火山和液体培养物。使用前,请将所有介质加热至室温。
  3. 用于HisTrap HP亲和色谱的裂解缓冲液
    50 mM Tris-Cl,pH 7.4
    2 M NaCl
    40 mM咪唑
    1 mg·ml -1无EDTA蛋白酶抑制剂混合物(Roche)
  4. 用于StrepTrap HP色谱的裂解缓冲液 50 mM Tris-Cl,pH 7.4
    2 M NaCl
    1 mg·ml -1无EDTA蛋白酶抑制剂混合物(Roche)
  5. Tris-盐缓冲液
    50 mM Tris碱
    2 M NaCl

    用浓盐酸滴定至pH 7.4或7.5
  6. 浓缩的检测缓冲液
    50 mM Tris-Cl,pH 7.5
    40 mM ATP
    250 mM DMSO
    20 mM MgCl 2 2/2 5 mM DTT
    2 M NaCl
  7. 2x SDS减缓缓冲液
    pH 6.8的100mM Tris-Cl缓冲液 4%(w / v)SDS
    20%(v / v)甘油

    0.6 mg·ml-1溴酚蓝 5%(v / v)β-巯基乙醇
  8. 12%SDS-PAGE凝胶
    1. 根据Bio-Rad在Mini-PROTEAN Tetra Handcast系统中浇铸凝胶,具有以下几点清晰度
    2. 分离凝胶:3.4ml去离子H 2 O,2.4ml 40%丙烯酰胺,2ml 1.5M Tris pH8.8,80μl10%SDS,80μl10%APS和8μlTEMED缓冲液>

    3. 在第一次聚合之前用去离子H 2 O将层积凝胶的顶层层叠
    4. 堆积凝胶:2.9ml去离子H 2 O,0.75ml 40%丙烯酰胺,1.25ml 0.5M Tris pH6.8,50μl10%SDS,10%APS和5μlTEMED
    5. 凝胶可以包裹在湿的Kimwipe和塑料包装中,并在4°C下储存长达1周
  9. 10倍运行缓冲区
    1. 将30g Tris碱,144g甘氨酸和10g SDS溶解在1L dH 2 O中。
    2. 不需要调整pH值。缓冲液的pH值应为8.3
    3. 在室温下储存并在使用前稀释至1x
  10. Transblot缓冲区
    500毫升0.2 M MES缓冲液pH 6
    1升100%甲醇
    8.5升去离子水
    在4°C储存
  11. 10x TBS(浓缩的Tris缓冲盐水)
    1. 将24克Tris碱和88克NaCl溶于900毫升蒸馏水中
    2. 用12 N HCl将pH调节至7.6

    3. 加入蒸馏水至最终体积为1升
  12. 含0.1%吐温20(TBST)的Tris缓冲盐水
    将100ml TBS 10x与900ml蒸馏H 2 O和1ml吐温20混合。

致谢

这项工作是由美国能源部,基础能源科学办公室,化学科学部,地球科学和生物科学部,物理生物科学计划(DE-FG02-05ER15650),美国国立卫生研究院|国家综合医学研究所(NIGMS)(NIH R01 GM57498-15)和美国农业部国家食品与农业研究所(Hatch 1005900)。该协议是根据Fu等人发表的以前的工作改编的。作者没有利益冲突或竞争利益声明。

参考

  1. Fu,X.,Adams,Z.,Liu,R.,Hepowit,N.L.,Wu,Y.,Bowmann,C.F.,Moskovitz,J。和Maupin-Furlow,J.A。(2017)。 甲硫氨酸亚砜还原酶A(MsrA)及其在古细菌泛素样蛋白修饰中的功能 a> MBio 8(5)。
  2. Glickman,M.H。和Ciechanover,A。(2002)。 泛素 - 蛋白酶体蛋白水解途径:为了构建而破坏。 Physiol Rev82(2):373-428。
  3. Hanahan,D.,Jessee,J.和Bloom,F.R。(1991)。 大肠杆菌和其他细菌的质粒转化。 Methods Enzymol 204:63-113。
  4. Hepowit,NL,de Vera,IM,Cao,S.,Fu,X.,Wu,Y.,Uthandi,S.,Chavarria,NE,Englert,M.,Su,D.,Sll,D.,Kojetin, DJ和Maupin-Furlow,JA(2016)。 机理性洞察蛋白质修饰和硫代动力学活动的noncanonical E1和相关泛素样蛋白质的古细菌。 FEBS J 283(19):3567-3586。
  5. Komander,D。和Rape,M。(2012)。 泛素编码
  6. Maupin-Furlow,J.A。(2014)。 原核泛素样蛋白修饰 Annu Rev Microbiol 68:155-175。
  7. Zhao,Q.,Liu,L。和Xie,Q。(2012)。 体外蛋白质泛素化分析 方法Mol Biol 876:163-172。
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引用:Fu, X., Adams, Z. and Maupin-Furlow, J. A. (2018). In vitro Analysis of Ubiquitin-like Protein Modification in Archaea. Bio-protocol 8(10): e2845. DOI: 10.21769/BioProtoc.2845.
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