Overexpression, purification, and characterization of recombinant Dictyostelium discoideum calcium-regulated 34,000-dalton F-actin bundling protein from Escherichia coli.
The Dictyostelium discoideum 34-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 0.4% 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 native D. discoideum 34-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-actin in 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.
Lim
R W
RW
Department of Cellular Biology, University of Georgia, Athens 30602, USA.
Fechheimer
M
M
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008840:Microfilament Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D000199:Actins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C052523:actin filament bundling proteins; D002118:Calcium