The BioLux Cypridina Luciferase Starter Kit contains a set of Cypridina Luciferase- expressing vectors and the reagents necessary for assaying the Cypridina Luciferase (CLuc) activity. The pSV40-CLuc Control Plasmid is a mammalian expression vector encoding the secreted Cypridina Luciferase from the Ostracod Cypridina noctiluca (1,2) under the control of the constitutive SV40 promoter. pSV40-CLuc can be used as a control to determine transfection efficiency. The pCLuc-Basic 2 Vector lacking promoter elements is a cloning vector for mammalian expression. pCLuc-Basic 2 contains a multiple cloning site (MCS) upstream of the CLuc coding sequence. In addition, the neomycin resistance gene in the pCLuc-Basic 2 cloning vector allows selection for stable integration of the plasmid into the mammalian cell genome.
Cypridina Luciferase is a 62 kDa protein with a native signal peptide at the N-terminus allowing it to be secreted from mammalian cells. This luciferase does not require ATP and catalyzes the oxidation of its luciferin substrate in a photochemical reaction (Figure 1). The substrate for Cypridina is different than coelenterazine, which is the common substrate of other marine luciferases including Renilla and Gaussia.
Properties of Cypridina Luciferase:
- Cypridina Luciferase is secreted from cells by virtue of its natural signal peptide and its luminescence can be measured from the supernatant of transfected cells. Therefore, cell lysis is not necessary (Figure 2).
- Secreted CLuc is a very stable protein. Because of this property, the activity measured from the supernatant reflects the amount of protein accumulated up to the time of sampling. Multiple samples can therefore be obtained from the same transfected cells (Figure 2).
- Secreted CLuc is thermally stable at 55°C (Figure 3A), which is the typical inactivation temperature of most viruses.
- Secreted CLuc remains active in the presence of β-mercaptoethanol, which is commonly present in the complete culture medium of mouse ES cells (Figure 3B).
- The CLuc assay is very sensitive, allowing detection of very small amounts of Cypridina Luciferase activity (Figure 4).
- Although the light reaction catalyzed by Cypridina has an initial emission half-life of approximately 5 minutes, light production continues to decay slowly, and is readily detectable 25 minutes after substrate addition (Figure 5).
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