Materials and Reagents

1.5 ml centrifuge tubes (Tarsons)
Dialysis tubing cellulose membrane (Sigma-Aldrich, catalog number: D9277 )
Amicon ultra centrifugal filters (EMD Millipore, catalog number: UFC900396 )
Cheese cloth
Freshly extracted or frozen chicken brain tissue
Saccharomyces cerevisiae yeast cells (BY4743 strain)
Freshly extracted liver tissue from chicken
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Sucrose (Sigma-Aldrich, catalog number: S7903 )
Sodium chloride (NaCl) (EMD Millipore, catalog number: 567440 )
Acetone (HiMedia Laboratories, catalog number: AS024 )
Lyticase (Sigma-Aldrich, catalog number: L4025 )
Protein A sepharose bead (GE Healthcare, catalog number: 17-0963-03 )
Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
Ammonium sulfate [(NH4)₂SO₄] (Sigma-Aldrich, catalog number: A2939 )
Coomassie Brilliant Blue R (Sigma-Aldrich, catalog number: B0149 )
Potassium chloride (KCl) (EMD Millipore, catalog number: 104936 )
Spermidine (Sigma-Aldrich, catalog number: S2626 )
Spermine (Sigma-Aldrich, catalog number: 85590 )
EDTA (Sigma-Aldrich, catalog number: EDS )
EGTA (Sigma-Aldrich, catalog number: E3889 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 7626 )
Disodium hydrogen phosphate (EMD Millipore, catalog number: 106586 )
Sodium dihydrogen phosphate (EMD Millipore, catalog number: 106370 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Glycerol (EMD Millipore, catalog number: 104093 )
Protease inhibitor cocktail (PIC) (Sigma-Aldrich, catalog number: P2714 )
Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
DTT (Sigma-Aldrich, catalog number: D9779 )
Sorbitol (Sigma-Aldrich, catalog number: S6021 )
Magnesium chloride hexahydrate (MgCl₂) (Sigma-Aldrich, catalog number: 63064 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
Trizma base (Sigma-Aldrich, catalog number: T6066 )
Hydroxyapatite resin (Sigma-Aldrich, catalog number: 289396 )
Sulfuric acid (H₂SO₄) (EMD Millipore, catalog number: 112080 )
Acrylamide (Sigma-Aldrich, catalog number: A8887 )
GLUD1 antibody (Sigma-Aldrich, catalog number: SAB2100932-50UG )
Note: This Product has been discontinued.
Solution 1 (see Recipes)
Hypotonic solution (see Recipes)
Sodium phosphate buffer (1 M), pH 6.8 (see Recipes)
HB buffer (see Recipes)
HAP buffer (see Recipes)
Potassium acetate solution (see Recipes)
Prespheroplasting buffer (see Recipes)
Spheroplasting buffer (see Recipes)
Wash buffer (see Recipes)
Lysis buffer (see Recipes)
Protein A sepharose beads (50% slurry) (see Recipes)
Yeast-protease resuspension buffer (see Recipes)
Protease dialysis buffer (see Recipes)
Reaction buffer

Yeast protease reaction buffer (see Recipes)
Chicken liver protease reaction buffer (see Recipes)

10% Triton X-100 solution (see Recipes)
6x SDS-PAGE loading dye (see Recipes)
10x SDS-PAGE running buffer (see Recipes)

Equipment

Homogenizer (Pro Scientific, model: Bio-Gen PRO200 homogenizer )
Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich, catalog number: P7734 )
Centrifuge (Beckman Coulter, models: Avanti^® J-E centrifuge-floor , Microfuge^® 22R centrifuge )
Sonicator (Boston Industries, model: Branson Digital Sonifier 250 & 102C Converter Ultrasonic Dismembrator )
Note: This product has been sold out.
Ultracentrifuge (Beckman Coulter, model: Optima^TM L-100K ultracentrifuge )
Bio-Rad electrophoresis power supply (Bio-Rad Laboratories, model: PowerPac^TM Basic Power Supply )
Bio-Rad SDS-PAGE running apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN tetra cell )
Shaker (Eppendorf, model: New Brunswick^TM Innova^® 44 )
Rotamer (Tarsons, catalog number: 3090 )
Heating block, Thermocell Cooling and heating Block (Bio-Equip, model: HB-202 )
FPLC cold cabinet (UniEquip, model: Unichromat 1500 )
Superose^TM 6, 10/300 GL chromatography column (GE Healthcare, catalog number: 17-5172-01 )

Procedure

Preparation of core histones
1. For protease assay, good quality of histones is required. We purified core histones from chicken brain tissue. To isolate histones from tissue requires preparation of nuclei as detailed below.
2. Purify core histones from isolated nuclei by hydroxyapatite affinity purification (HAP) method as described below:
Purification of H3 protease
1. Partial purification of H3-protease from Saccharomyces cerevisiae
2. Partial purification of H3-protease from chicken liver
Set up of in vitro histone H3 cleavage assay
1. Protease source: yeast (BY4743 strain)
2. Protease source: chicken
Visualization of core histones after assay
1. Stop the in vitro cleavage reaction by the addition of SDS-PAGE loading dye as mentioned earlier.
2. Incubate the samples at 95 °C for 5 min.
3. Examine the cleavage of histone H3 by running 18% SDS-PAGE as shown in Figure 3.
4. Visualize the bands of core histones by staining with Coomassie blue staining.
  
  Figure 3. In vitro histone H3 cleavage assay with core histones. A. In vitro activity assay with yeast protease. B. In vitro activity assay with chicken liver protease. Out of four core histones, only histone H3 get cleaved as protease is specific for histone H3. ‘-’ and ‘+’ indicate absence and presence of H3 protease respectively.
  Note: The in vitro histone H3 cleavage assay works best when proteases are freshly prepared. The core histone should be stored in small aliquots at -80 °C. Repeated freeze-thawing of both histones and proteases should be avoided.

Recipes

Solution 1
0.34 M sucrose
15 mM Tris-Cl, pH 7.5
15 mM NaCl
60 mM KCl
0.5 mM spermidine
0.15 mM spermine
2 mM EDTA
0.5 mM EGTA
15 mM β-mercaptoethanol
0.2 mM PMSF (added before use)
Hypotonic solution
10 mM Tris-Cl, pH 7.5
15 mM β-mercaptoethanol
2 mM PMSF
Sodium phosphate buffer (1 M), pH 6.8 (100 ml)
49 ml of 1 M disodium hydrogen phosphate
51 ml of sodium dihydrogen phosphate
HB buffer
20 mM HEPES, pH 7.5
0.4M NaCl
1 mM EDTA
10% glycerol
0.1% NP-40
1 mM β-ME
PIC (Protease inhibitor cocktail) (added before use)
HAP buffer
50 mM sodium phosphate buffer
1 mM β-ME
PIC (Protease inhibitor cocktail) (added before use)
Potassium acetate solution
2 g potassium acetate dissolved in 100 ml water
Prespheroplasting buffer
100 mM Tris-Cl, pH 8.8
10 mM DTT
Spheroplasting buffer
50 mM Tris-Cl, pH 7.5
0.6 M sorbitol
10 mM DTT
Wash buffer
100 mM KCl
50 mM HEPES-KOH, pH 7.5
2.5 mM MgCl₂
0.4 M sorbitol
Lysis buffer
20 mM Tris-Cl, pH 8.5
200 mM KCl
25 mM EDTA
1% (v/v) Triton X-100
Protein A sepharose beads (50% slurry)
Take 500 µl protein A sepharose beads in 1.5 ml Eppendorf tube
Centrifuge it at 4 °C, 800 x g for 5 min
Discard supernatant
Add of 1 ml of lysis buffer to beads
Centrifuge it again and discard the supernatant
Repeat it two more time
Finally, resuspend the bead pellet in 500 µl of lysis buffer to obtain 50% slurry
Yeast-protease resuspension buffer
20 mM Tris-Cl, pH 8.5
200 mM KCl
25 mM EDTA
Protease dialysis buffer
25 mM Tris-Cl, pH 7.5
100 mM NaCl
10% glycerol
1 mM β-mercaptoethanol
0.2 mM EDTA
Reaction buffer

Yeast protease reaction buffer
25 mM Tris-Cl, pH 7.5
150 mM NaCl
10% glycerol
2 mM β-mercaptoethanol
0.1 mM EDTA
Chicken liver protease reaction buffer
10 mM HEPES, pH 5.5
100 mM NaCl
1 mM β-mercaptoethanol
0.2 mM EDTA
10% glycerol

10% Triton X-100 solution
10 ml of Triton X-100 from stock (100%)
90 ml water
Solution mixed well through rotation
10% solution
Stored at 4 °C
6x SDS-PAGE loading dye
2.5 ml of 4x Tris-Cl, pH 6.8
3 ml glycerol
1 g SDS, 0.5 ml
1.2 mg bromophenol blue
Add water to make final volume to 10 ml
10x SDS-PAGE running buffer
30.0 g of Trizma base
10.0 g of SDS
144.0 g of glycine
Dissolve in 1,000 ml of H₂O
Dilute 10x buffer to 1x before use

Acknowledgments

This work was supported by the Department of Biotechnology (Government of India), Department of Science and Technology (Government of India) to RST. Short version of this protocol was published in Chauhan et al., 2016, Chauhan and Tomar, 2016; Azad and Tomar, 2016; Mandal et al., 2014; Mandal et al., 2013; Mandal et al., 2012. CSIR is acknowledged for providing fellowship support to Sakshi. Authors also acknowledge members of RST lab for their comments on this work.

References

Azad, G. K. and Tomar, R. S. (2014). Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes. Mol Biol Rep 41(5): 2717-2730.
Azad, G. K. and Tomar, R. S. (2016). Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae. Yeast 33(6): 217-226.
Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21(3): 381-395.
Chauhan, S., Mandal, P. and Tomar, R. S. (2016). Biochemical analysis reveals the multifactorial mechanism of histone H3 clipping by chicken liver histone H3 protease. Biochemistry 55(38): 5464-5482.
Chauhan, S. and Tomar, R. S. (2016). Efficient expression and purification of biologically active human cystatin proteins. Protein Expr Purif 118: 10-17.
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Mandal, P., Azad, G. K. and Tomar, R. S. (2012). Identification of a novel histone H3 specific protease activity in nuclei of chicken liver. Biochem Biophys Res Commun 421(2): 261-267.
Mandal, P., Chauhan, S. and Tomar, R. S. (2014). H3 clipping activity of glutamate dehydrogenase is regulated by stefin B and chromatin structure. FEBS J 281(23): 5292-5308.
Mandal, P., Verma, N., Chauhan, S. and Tomar, R. S. (2013). Unexpected histone H3 tail-clipping activity of glutamate dehydrogenase. J Biol Chem 288(26): 18743-18757.
Workman, J. L., Taylor, I. C., Kingston, R. E. and Roeder, R. G. (1991). Control of class II gene transcription during in vitro nucleosome assembly. Methods Cell Biol 35: 419-447.