Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin.
The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.
Inouye
S
S
Basic Research Laboratory, Chisso Corporation, Kanagawa.
Aoyama
S
S
Miyata
T
T
Tsuji
F I
FI
Sakaki
Y
Y
D000331:Aequorin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001059:Apoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002848:Chromatography, DEAE-Cellulose; D003063:Cnidaria / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D007525:Isoelectric Focusing; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000096:biosynthesis*; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D037401:Scyphozoa / Q000378:metabolism*
D000596:Amino Acids; D001059:Apoproteins; D002135:Calcium-Binding Proteins; D004269:DNA, Bacterial; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin