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

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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)146-150
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume383
Issue number1
DOIs
Publication statusPublished - 2009 May 22
Externally publishedYes

Fingerprint

Gelsolin
Affinity chromatography
Affinity Chromatography
Purification
Actins
Myosin Type V
Actin Capping Proteins
Baculoviridae
Actin Cytoskeleton
HeLa Cells
Polymerization
Sepharose
Insects
Adenosine Triphosphatases
Chelation
Escherichia coli
Salts
Assays
Genes

Keywords

  • Actin purification
  • Actin-binding protein
  • Baculovirus
  • Gelsolin
  • Recombinant actin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin. / Ohki, Takashi; Ohno, Chikanori; Oyama, Kotaro; Mikhailenko, Sergey; Ishiwata, Shin'ichi.

In: Biochemical and Biophysical Research Communications, Vol. 383, No. 1, 22.05.2009, p. 146-150.

Research output: Contribution to journalArticle

Ohki, Takashi ; Ohno, Chikanori ; Oyama, Kotaro ; Mikhailenko, Sergey ; Ishiwata, Shin'ichi. / Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin. In: Biochemical and Biophysical Research Communications. 2009 ; Vol. 383, No. 1. pp. 146-150.
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