Overexpression, Purification, and Characterization of RecombinantDictyostelium discoideumCalcium-Regulated 34,000-Dalton F-Actin Bundling Protein fromEscherichia coli

Abstract

TheDictyostelium discoideum34-kDa protein is an F-actin bundling protein that demonstrates diverse distributions in the cell during cell shape changes and cell movement. The protein is expressed at a very low level in the amoeba, just 0.04% of the total cell protein. This presents a challenging problem when purifying sufficient protein for structural and biochemical studies. The purification procedure is lengthy and yields only a few milligrams of protein. An alternative protein expression system, that of the bacterial T7 expression system, was used to produce large quantities of recombinant 34-kDa protein (r34-kDa). The soluble r34-kDa protein constitutes up to a quarter of the total bacterial protein, and was purified to homogeneity by a modification of the purification procedure for the nativeD. discoideum34-kDa protein (N34-kDa). The r34-kDa possesses all the same functional characteristics as the N34-kDa protein with respect to its interactions with F-actinin vitro:it bound to and cross-linked F-actin, mediated F-actin bundle formation, directly bound calcium, and demonstrated calcium-sensitive F-actin binding activities.