Membrane Targeting of Firefly Luciferase: A New Bioluminescent Reporter Gene

• Published in: Analytical biochemistry 1994-03, Vol.217 (2), p.333-335
• Main Authors: Badia, E., Duchesne, M.J., Pons, M., Nicolas, J.C.
• Format: Article
• Language: English
• Subjects: Animals; Avian Sarcoma Viruses - genetics; Base Sequence; Biological and medical sciences; Coleoptera - enzymology; Diverse techniques; Estradiol - pharmacology; Fundamental and applied biological sciences. Psychology; Luciferases - genetics; Luciferases - metabolism; Luminescent Measurements; Molecular and cellular biology; Molecular Sequence Data; Photinus pyralis; Plasmids - genetics; Promoter Regions, Genetic - drug effects; Promoter Regions, Genetic - genetics; Receptors, Serotonin - genetics; Receptors, Serotonin, 5-HT1; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Transfection; Vitellogenins - genetics; Xenopus laevis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1994.1129

In the last few years, the study of promoter activation has greatly expanded, often using promoter constructs coupled to luciferase genes. It soon appeared interesting to develop luciferase chimeric genes targeted to various cellular compartments. To our knowledge, no attempt has been made to fuse luciferase to a membrane receptor protein. This would offer the possibility of easily separating modified and wild-type luciferase proteins expressed by two different expression vectors in the same cell. For this purpose we fused the luciferase from Photinus pyralis downstream of the second external arm of the HT1a receptor which belongs to the well-known family of G-linked protein receptors. In our construct, the HT1a-Luc chimeric protein gene as well as the wild luciferase protein gene was placed under control of the estradiol-inducible promoter of the Xenopus vitellogenin gene or under control of the strong Rous sarcoma virus promoter. We show that the fusion protein is enzymatically active, associated to the insoluble fraction of the cell, and easily separable from the soluble fraction. It may be associated with the corresponding soluble reporter enzyme for monitoring two different responses in the same cell. It could be used for normalization in transient transfection experiments. Construction of the pVit-tk-HT1a-Luc and pRSV-HT1a-Luc plasmids. Plasmids were constructed according to standard protocols for recombinant DNA technology. The pVit-tk-luc plasmid, which contains the firefly luciferase coding sequence from P. pyralis under control of the vitellogenin gene promoter (Vit), has been described elsewhere. In this plasmid, the 5'-flanking region of Vit contains an estrogen-responsive element which is inserted in front of the herpes simplex virus promoter for thymidine kinase (tk). This promoter is therefore inducible by estrogens. The pRSV-luc plasmid was constructed by exchanging the Vit-tk promoter with the strong noninducible Rous sarcoma virus promoter (RSV) in order to obtain the membrane-targeted luciferase.