title: High-level expression and purification of apoaequorin.
abstract: A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.
authors.affiliation: Yokohama Research Center, Chisso Corporation, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Inouye
authors.forename: S
authors.initials: S
authors.lastname: Zenno
authors.forename: Y
authors.initials: Y
authors.lastname: Sakaki
authors.forename: F I
authors.initials: FI
authors.lastname: Tsuji
mesh_terms: D000331:Aequorin / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics* / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins; D037401:Scyphozoa / Q000235:genetics
chemical_list: D001059:Apoproteins; D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin