A Novel Streptavidin–luciferase Fusion Protein: Preparation,Properties and Application in Hybridization Analysis of DNA

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.     

Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.     

Stabilization of <Emphasis Type="Italic">Luciola mingrelica</Emphasis> firefly luciferase by genetic engineering methods

The results of Luciola mingrelica firefly luciferase stabilization by genetic engineering methods are reviewed. The Cys62, Cys146, and Cys164 to Ser mutant enzymes with an enhanced thermostability and lower sensitivity to dithiothreitol were obtained by site-directed mutagenesis. The double mutant G216N, A217L was obtained, which displayed a higher thermostability and resistance to DMSO in comparison with WT luciferase. Random mutagenesis of the gene region encoding residues 1–225 and subsequent screening of the mutants resulted in the production of the mutant MT8 with a higher thermostability, as well as mutants MT3 and MT4 with higher resistance to dimethyl sulfoxide. The mutant 4TS was obtained by the method of directed evolution of the gene site encoding residues 130–390, which was shown to contain eight replacements after four cycles of mutagenesis and had two-fold higher specific activity, eight-fold lower K _m value for ATP, and stability at 42°C, which was 65-fold higher that of WT luciferase. The stabilization mechanism of this mutant is discussed.     

Synthesis and application of firefly luciferase antibody conjugates in a bioluminescent immunoassay of Salmonella cells

A method has been developed for synthesizing stable and highly active conjugates of firefly luciferase with antibodies using the heterobifunctional coupling agent N-succinimidyl 3-(2-pyridyldithio) propionate linking NH₂ groups of antibodies with free SH groups of thermostable Luciola mingrelica firefly luciferase (Luc). A novel specific matrix for sorption of Salmonella cells has been designed that includes monodisperse polystyrene microparticles (φ = 240 nm) coated with Pluronic F108-PDS, which are then covalently linked to monoclonal antibodies against Salmonella cells (Sal). The Luc-Sal conjugates are used as detecting agents for bioluminescent immunoassay of Salmonella cells. The use of the novel matrix and highly active conjugates allow for a ～100-fold increase in the method’s sensitivity compared with the standard sandwich assay.     

Emitter as an Intramolecular Probe in a Luciferase Active Center

Moscow University Chemistry Bulletin - The distinctive features of firefly luciferase bioluminescence are complex changes in the shape of the spectra and λmax of bioluminescence with varying...     

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λ_max.em = 590 nm (RedLuc) and the “green” mutant with λ_max.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL^?1.     

Controlling BCG Vaccine’s Cell Viability in the Process of Its Production by an Bioluminescent ATP Assay

Moscow University Chemistry Bulletin - The Bacille Calmette–Guerine (BCG) vaccine, which is based on a live strain of Mycobacterium bovis BCG is widely used for the immunoprophylaxis of...     

Immobilized bacterial luciferase and its applications

This review discusses the properties of the bioluminescent bacterial system as well as the methods for immobilization of bacterial luciferases and for their co-immobilization with other enzymes. The analytical systems using immobilized bacterial luciferases and their applications in analytical biochemistry and biotechnology have been described.     

Relationship between the structure of the protein globule and bioluminescence spectra of firefly luciferase

Bioluminescence spectra for various native and mutant luciferases from fireflies and beetles were analyzed in the light of the known theoretical concepts on the influence of the microenvironment of the emitter on its emission spectra. The mechanism for the explanation of the nature of changing bioluminescence spectra for natural and artificial mutations of the amino acid residues in the protein globule of luciferases was proposed.     

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin‐binding domain and streptavidin, with proteins A and G, antibodies, with DNA‐ and RNA‐binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase‐based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET‐based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein–protein interactions, assaying of metabolites involved in cell communication and cell signaling.