A Large-Scale Purification of Recombinant Histone H1.5 from Escherichia coli

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-pressur...

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Bibliographic Details
Published in:Protein expression and purification 2001-10, Vol.23 (1), p.38-44
Main Authors: Pyo, Sang-Hyun, Lee, Jae-Hyun, Park, Heung-Bok, Hong, Seung-Suh, Kim, Jin-Hyun
Format: Article
Language:English
Subjects:
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Electrophoresis, Polyacrylamide Gel
Endotoxins - analysis
Escherichia coli - chemistry
Escherichia coli - genetics
Histones - chemistry
Histones - isolation & purification
Humans
Hydroxyapatites
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Ultrafiltration
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1471