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 Scientific^TM, 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 Scientific^TM, 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 Scientific^TM, 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 Scientific^TM, 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 Scientific^TM, 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 Scientific^TM, 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 P2_rrnA constitutive promoter) (Fu et al., 2017) or pJAM1209 (a plasmid encoding his₆-ubaA under control of the P2_rrnA 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 Scientific^TM, 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, Invitrogen^TM, 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, Bacto^TM, catalog number: 211705 )
    
72. BBL dehydrated culture media additive: Yeast extract (BD, BBL^TM, 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 Scientific^TM, 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 Scientific^TM, catalog number: 76981 ) and Sorvall SS-34 fixed angle rotor (Thermo Fisher Scientific, Thermo Scientific^TM, 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 A_{562 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 Brunswick^TM, 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 Brunswick^TM, 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