The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coil 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 alpha-fetoprotein as a model analyte was 0.02-100 ng/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. (C) 2011 Elsevier Inc. All rights reserved.