title: A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.
abstract: Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C.
authors.affiliation: Department of Genome Sciences, The John Curtin School of Medical Research, Building 131, Garran Road, The Australian National University, Canberra, ACT, 2601, Australia. Electronic address: daniel.ryan@anu.edu.au.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Ryan
authors.affiliation: Department of Genome Sciences, The John Curtin School of Medical Research, Building 131, Garran Road, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Tremethick
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase; D006639:Histidine; D006657:Histones / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: C471213:His-His-His-His-His-His; D006657:Histones; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D005982:Glutathione Transferase
keywords: Affinity purification; Chromatin; Linker histones; Nucleosomes; Recombinant protein; Rhamnose