title: Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: expression, purification and its application for bioluminescent immunoassays.
abstract: The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02-100ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme.
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
authors.forename: Yuiko
authors.initials: Y
authors.lastname: Sahara-Miura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D033342:Copepoda / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008163:Luminescent Measurements / Q000379:methods*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D008156:Luciferases; D003545:Cysteine