The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin

Recombinant apoaequorin expressed in the periplasmic space of Escherichia c oli cells was regenerated into aequorin and extracted from the cells, simul taneously, using a buffer that contained coelenterazine. Due to the mild ex traction 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 ae quorin was pure, based on various data, including HPLC analysis and light-e mitting activity. The yield of purified aequorin was 25-35 mg from 600 mi o f culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells. (C) 1999 Academic Press.