Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coil cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3 mg of His-Cys4-aequorin from 2 L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-acitivated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for alpha-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01-100 ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin. (C) 2012 Elsevier Inc. All rights reserved.