Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

Takashi Ohki, Chikanori Ohno, Kotaro Oyama, Sergey V. Mikhailenko, Shin'ichi Ishiwata*

*この研究の対応する著者

研究成果: Article査読

19 被引用数 (Scopus)

抄録

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca2+ and incubated overnight at 4 °C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca2+-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.

本文言語English
ページ(範囲)146-150
ページ数5
ジャーナルBiochemical and Biophysical Research Communications
383
1
DOI
出版ステータスPublished - 2009 5月 22
外部発表はい

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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