title: Purification of histidine-tagged aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays.
abstract: 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 40.3mg of His-Cys4-aequorin from 2L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-activated 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.
authors.affiliation: Yokohama Research Center, JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan. sinouye@jnc-corp.co.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inouye
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sato
mesh_terms: D000331:Aequorin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods; D008301:Maleimides / Q000737:chemistry; D008956:Models, Chemical; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000032:analysis
chemical_list: D008301:Maleimides; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; C043592:maleimide; D006639:Histidine; D000331:Aequorin; D001710:Biotin; D019809:Streptavidin; D003545:Cysteine