title: The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.
abstract: Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.
authors.affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
authors.forename: O
authors.initials: O
authors.lastname: Shimomura
authors.forename: S
authors.initials: S
authors.lastname: Inouye
mesh_terms: D000331:Aequorin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin