An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis

AbstractPrevious 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

Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors

Total synthesis of a gene for bovine rhodopsin

To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5'-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene