Purification of Histone Chaperones and Histones

sNASP was expressed in bacteria as an N-terminal (His)₆ fusion construct using Ni-NTA affinity resin (GE Healthcare) and was further purified by ion-exchange and gel filtration chromatography after cleavage of the (His)₆ fusion by TEV protease, as described previously (25 (link)). sNASP mutants, truncations and the budding yeast homolog Hif1 were expressed and purified using the same method. N-terminal tagged GST-ASF1A was expressed in bacteria and purified over a Glutathione Sepharose resin (GE Healthcare), the GST tagged removed with Precision Protease cleavage, and further purified using anion exchange and gel filtration chromatography. Mb13 was expressed as a (His)₆, Avitag fusion in bacteria, and after cleavage of the (His)₆ fusion, was further purified by ion exchange and size exclusion chromatography. Full length Xenopus histones H3 and H4 were purified and refolded as described previously to form the H3–H4 dimer/tetramer (33 (link)). For reconstitution of monomeric histones with chaperones, H3 and H4 were dialyzed to water and then added directly to the chaperone. MBP–H3 (116–135) and MBP-mb1 were expressed in the same way as sNASP and purified over Dextrin Sepharose (GE Healthcare) in 20 mM HEPES–KOH pH 7.5 and 0.5 M sodium chloride, eluted in the same buffer supplemented with 10 mM maltose. Maltose was removed by dialysis prior to storage at –80°C.