Synthesis of mammalian profilin in Escherichia coli and its characterization.
Profilin is a G-actin binding protein that may have a role in controlling the ratio of G/F actin within the cell. To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfected Escherichia coli with a plasmid containing a full-length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly-L-proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G-75 and DEAE A-50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kd of approximately 2 microM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure-function studies involving mutant profilins altered by site-directed mutagenesis.
Babcock
G
G
Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242.
Rubenstein
P A
PA
D000199:Actins / Q000378:metabolism; D000818:Animals; D001704:Biopolymers; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003285:Contractile Proteins; D004926:Escherichia coli / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D008027:Light; D008840:Microfilament Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D051304:Profilins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012542:Scattering, Radiation
D000199:Actins; D001704:Biopolymers; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011994:Recombinant Proteins