An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis.
Previous studies by Papermaster and coworkers introduced the use of rhodopsin-green fluorescent protein (rho-GFP) fusion proteins in the construction of transgenic Xenopus laevis with retinal rod photoreceptor cell-specific transgene expression [Moritz et al., J. Biol. Chem. 276 (2001) 28242-28251]. These pioneering studies have helped to develop the Xenopus system not only for use in the investigation of rhodopsin biosynthesis and targeting, but for studies of the phototransduction cascade as well. However, the rho-GFP fusion protein used in the earlier work had only 50% of the specific activity of wild-type rhodopsin for activation of transducin and only 10% of the activity of wild-type in rhodopsin kinase assays. While not a problem for the biosynthesis studies, this does present a problem for investigation of the phototransduction cascade. We report here an improved rhodopsin/EGFP fusion protein in which placement of the EGFP domain at the C-terminus of rhodopsin results in wild-type activity for activation of transducin, wild-type ability to serve as a substrate for rhodopsin kinase, and wild-type localization of the protein to the rod photoreceptor cell outer segment in transgenic X. laevis.
Jin
Shengnan
S
Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, MA 02454, USA.
McKee
Timothy D
TD
Oprian
Daniel D
DD
D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012160:Retina / Q000737:chemistry / Q000166:cytology; D012243:Rhodopsin / Q000235:genetics*; D015293:Transducin / Q000378:metabolism; D014785:Vision, Ocular; D014982:Xenopus laevis / Q000235:genetics*
D005136:Eye Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D015293:Transducin