Purification Of Cytosolic Actin By Affinity Chromatography Using C-terminal Half Of Gelsolin

Abstract

Actin filaments in living cells undergo continuous dynamic turnover and remodeling. These processes involve polymerization, depolymerization, severing, capping, and branching of actin filaments through the interaction with a vast array of actin binding proteins. Cytoplasmic actin had previously been purified by the affinity chromatography using the immobilized DNase-I. which binds to G-actin with high affinity (K(d) = 0.05 nM). After being eluted from a DNase-I column, actin had to be exposed to high concentrations of a denaturant, such as 10 M formamide or 3 M guanidine-hydrochloride. We introduced a new method of the cytosolic actin purification, based on the affinity chromatography using a carboxyl-terminal half of gelsolin (G4-G6), which is an actin filament severing and capping protein, without the use of a denaturant. G4-G6 strongly binds to G-actin (K(d) = 30 nM) and has the actin-nucleating activity. His-tagged G4-G6 (His-G4-G6) was expressed in Escherichia coli and purified by Ni-affinity chromatography. When His-G4-G6 was added to a lysate of HeLa cells or insect cells infected with a baculovirus, expressing the beta-actin, in the presence of calcium and incubated overnight at 4 degrees centigrade, His-G4-G6 bound to actin with a 1:1 stoichiometry. His-G4-G6-actin complex was purified with Ni-agarose resin, and only actin was eluted from Ni-column by calcium chelation. To examine whether the purified actins were functional, we measured the polymerizability of actins and the velocity of actin filaments in an in vitro motility assay on myosin V. At this meeting, we report the properties of purified actins.