ABSTRACT
The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%)>RLase (8.0%)>RLase-547 (0.73%)>19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases.
Subject(s)
Copepoda/enzymology , Luciferases, Renilla/genetics , Luciferases, Renilla/isolation & purification , Renilla/enzymology , Animals , Catalysis , Escherichia coli , Imidazoles/chemistry , Luciferases, Renilla/biosynthesis , Luciferases, Renilla/chemistry , Luminescent Measurements , Pyrazines/chemistry , Substrate SpecificityABSTRACT
The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.
Subject(s)
Annexin A5/metabolism , Apoptosis , Luciferases, Renilla/metabolism , Molecular Probes/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Humans , Jurkat Cells , Luciferases, Renilla/genetics , Luciferases, Renilla/isolation & purification , Molecular Probes/genetics , Molecular Probes/isolation & purification , Neutrophils/cytology , Neutrophils/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Staining and LabelingABSTRACT
Analyzing the putative interaction partners of an individual protein is one approach to elucidate its function. In the LuMPIS protocol, bait and prey proteins are expressed with N-terminal maltose binding protein (MBP)- and eGFP-luciferase (eGFP-luc) tags, respectively. Positive protein-protein interactions (PPIs) can be detected after pull-down of the MBP-tagged prey protein using amylose beads followed by the bioluminescence detection of the bound eGFP-luc-tagged bait protein. The LuMPIS technology offers the following advantages: the PPIs are detected in the mammalian cell context, the use of two long protein tags (i.e., MBP and eGFP-luc) increases the expression levels of genes whose gene expression levels are known to be frequently impaired, the use of amylose beads for pull-down is much more economic as compared to sepharose beads in combination with monoclonal antibodies and finally, the use of an eGFP-luc-tag enables the qualitative control of transfection efficiencies by fluorescence microscopy prior to starting the assay.
