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A Novel Streptavidin–luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA
Author(s) -
Smirnova Daria V.,
Rubtsova Maya Y.,
Grigorenko Vitaly G.,
Ugarova Natalia N.
Publication year - 2016
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.12666
Subject(s) - streptavidin , fusion protein , fusion , dna , luciferase , chemistry , computational biology , microbiology and biotechnology , biophysics , biochemistry , biology , biotin , recombinant dna , gene , transfection , linguistics , philosophy
A streptavidin–luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin–luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin‐binding activities. It was shown that fusion has the same K m values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10 −18 –10 −13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin–streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin–luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.