Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays

Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli 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 α-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.

► Histidine-tagged aequorin with a reactive cysteine residue was highly purified. ► Aequorin-streptavidin conjugate was applied to the bioluminescenct sandwich immunoassay. ► The measurable range of α-fetoprotein was 0.01∼100 ng/ml. ► The sensitivity was similar to aequorin-labeled antibody and biotinylated aequorin.