Recombinant oligonucleosomes

Part1 Histones purification

1.      The open reading frames of histone H2A, H2B, H4, wildtype or mutant histone H3.3 were sub-cloned into the pET28a vector and the plasmids were transformed into E. coli strain Rosetta (DE3).

2.      The transformants were grown at 37°C to OD₆₀₀ of 0.8 and proteins expression were induced by adding 0.2 mM IPTG (final concentration).

3.      After further culturing at 37 °C for 2 hours, cells were harvested by centrifugation at 5000 rpm for 10 min, and lysed with 35 ml lysis buffer (for 1 L bacteria) containing 50 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA and 1 mM PMSF.

4.      The bacterial lysates were then sonicated at 200W “power”, 30s “on”, 30s “off” for 20 “cycles”, and then centrifugated at 12,000 rpm for 10 min at 4 °C.

5.      The pellets were washed three times with 35 ml lysis buffer plus 1% TritonX-100 and 35 ml three times with lysis buffer, and centrifugated at 12,000 rpm for 10 min at 4 °C.

6.      The pellets were resuspended in unfolding buffer containing 7M guanidinium-HCl, 20 mM Tris-HCl pH7.5, 10 mM DTT and mixed gently for 1 hour at room temperature.

7.      The purified histones were quantitated by 15% SDS-PAGE followed by Coomassie Blue staining.

Part2 Histone octamers

1.      Histone octamers were obtained by mixing the four unfolded recombinant histones isolated as described above in equimolar amounts with approximately 4mg of total protein in about 1ml volume.

2.      Then the mixtures were dialyzed at 4℃ against 2 L of refolding buffer (10 mM Tris-HCl pH7.5, 2 M NaCl, 1 mM EDTA and 5 mM β-Mercaptoethanol) with at least three buffer changes. Either the second or third dialysis step were performed overnight.

3.      The samples were centrifuged to remove any precipitates and proteins were concentrated to 250 μl with a Millipore microconcentrator (10-kDacutoff, Millipore, UFV4BGC00). Then the samples were loaded onto a HiLoad Superdex 200 HR 10/30 column equilibrated with refolding buffer.

4.      Recombinant core histone octamers were eluted with a relative molecular mass of 100kDa, and were analyzed by 15% SDS-PAGE followed by Coomassie Blue staining.

Part3 Recombinant oligonucleosomes

1.      Recombinant oligonucleosomes were obtained by mixing the recombinant core histone octamers and plasmid containing tandem 147 bp DNA fragments in a mass ratio 1:1.2 at room temperature for 15 min.

2.      Then the mixtures were dialyzed at 4℃ against 1L of TEN buffers containing 10mM Tris-HCl pH7.5, 1 mM EDTA, 5 mM β-Mercaptoethanol and different concentrations of NaCl- 1.4 M, 1.2 M, 1.0 M, 0.8 M, 0.6 M.

3.      The mixtures were dialyzed at 4℃ against 1L of TE buffer containing 10 mM Tris-HCl pH7.5 and 1 mM EDTA overnight.

4.      The recombinant oligonucleosomes were quantitated by 15% SDS-PAGE followed by Coomassie Blue staining.