Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin.
Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, gave light with Ca2+. A method is described for the preparation of highly pure aequorin. The aequorin was stable in solution for approximately 10 days at 4 degrees C and pH 7.6, and then it gradually lost activity with a half-life of about 20 days until it was almost completely inactive on day 30.
Masuda
Hiromi
H
Department of Biochemistry, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Takenaka
Yasuhiro
Y
Shikamoto
Yasuo
Y
Kagawa
Masayuki
M
Mizuno
Hiroshi
H
Tsuji
Frederick I
FI
D000331:Aequorin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013997:Time Factors
D001059:Apoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin; D002118:Calcium