title: Expression, purification, and structural characterization of human histone H4.
abstract: Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry.
authors.affiliation: Department of Biophysical Sciences and Technologies M.&O., University of Genoa, Genoa, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Vergani
authors.forename: Fabio
authors.initials: F
authors.lastname: Canneva
authors.forename: Paola
authors.initials: P
authors.lastname: Ghisellini
authors.forename: Claudio
authors.initials: C
authors.lastname: Nicolini
mesh_terms: D002942:Circular Dichroism; D006657:Histones / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D006657:Histones