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KMID : 1094720080130060772
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Biotechnology and Bioprocess Engineering
2008 Volume.13 No. 6 p.772 ~ p.777
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Purification and characterization of histone H1 variants from human placenta
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Pyo Sang-Hyun
Lee Jae-Heung
Lee Yeune-Hee
Yoon Jong-Won
Kim Jin-Hyun
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Abstract
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Three histone H1 variants were extracted from human placental tissue in a single process using a high-salt buffer solution, and purified by ion exchange, hydroxyapatite, and reversed-phase chromatography. In the first chromatographic step, a cation exchanger resin, SP-Sepharose FF, was used to remove impurities having molecular weights higher than those of histones. In the second chromatographic step, hydroxyapatite resin was used to remove impurities with relatively low molecular weights. A second round of cation exchange chromatography using high-grade HS POROS resin resulted in two main fractions, each of which appeared as a single band following SDS-PAGE. The first fraction showed a single peak in RP-HPLC, while the second fraction showed two main peaks. These three peaks were further separated and polished by semi-preparative RP-HPLC, and their molecular masses and sequences were determined using MALDI-TOF-MS and N-terminal amino acid sequencing, respectively. The sequences and masses of these three variants corresponded with those of histones H1.2, H1.4, and H1.5. Moreover, all three purified histone subtypes demonstrated cytotoxicity in an MTT assay.
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KEYWORD
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histone H1 variants, purification, characterization, cytotoxicity, human placenta
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