title: A large-scale purification of recombinant histone H1.5 from Escherichia coli.
abstract: An Escherichia coli expression system has been constructed for production of biologically active recombinant histone H1.5. A process of fermentation and purification method at a large scale has been developed. Recombinant histone H1.5 was released from the high density cultured cells by high-pressure homogenization. For an efficient removal of cell debris and partial purification of basic histone H1.5 in a single step, the whole cell lysates were directly loaded onto an expanded bed column packed with the strong cation exchanger (Streamline SP). Complete removal of various impurities was achieved by a combination of hydroxyapatite chromatography and the following cation exchange chromatography with high grade strong cation exchanger (POROS 20 HS), and finally endotoxins were removed by ultrafiltration using a 100-kDa cut-off membrane, which gave the level of endotoxin below 0.5 EU/mg. The molecular mass of the recombinant histone H1.5 analyzed by MALDI-TOF-MS, and the N-terminal amino acid sequences were in good agreement with the authentic histone H1.5. The whole process gave highly purified recombinant histone H1.5 at a high yield, compared to the conventional process.
authors.affiliation: Samyang Genex Biotech Research Institute, Taejeon, 305-348, South Korea.
authors.forename: S H
authors.initials: SH
authors.lastname: Pyo
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: H B
authors.initials: HB
authors.lastname: Park
authors.forename: S S
authors.initials: SS
authors.lastname: Hong
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics*; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006882:Hydroxyapatites; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014462:Ultrafiltration
chemical_list: D004731:Endotoxins; D006657:Histones; D006882:Hydroxyapatites; D011994:Recombinant Proteins