No abstract

Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human P,-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350000 human receptorskell. The number of receptors did not change with cell density or age of culture. The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. Coupling of the receptors to endogenous GTP-binding proteins is demonstrated by hormone-dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells. Western-blot analysis revealed that the endogenous GTP-binding protein appears to be of the heterotrimeric type. Antibodies raised against the mammalian a subunit of stimulatory GTP-binding proteins cross-react with the insect a subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The p subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the Cterminal amino acids of the mammalian / 3 subunit of GTP-binding proteins, but is about =2 kDa larger than that of the P subunit of GTP-binding proteins from bovine brain. Exposure of the transformed insect cells to L-isoproterenol rapidly induces uncoupling and internalization of 30 % of the heterologously expressed receptors. In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remaining 70% of the receptors.Mammalian P-adrenergic receptors CB,AR) have been introduced into several heterologous expression systems for extensive pharmacological and biochemical analysis, including Escherichia coli (Marullo et al., 1989(Marullo et al., ,1990, yeast (King et al., 1990), Xenopus oocytes (Frielle et al., 1988;Kobilka et al., 1988), several mammalian cell lines Fraser et al., 1987;Hen et al., 1989;Lohse, 1992;Rands et al., 1990;Wang et al., 1989) and insect cells, using the baculovirus expression system (George et al., 1989;Reiliinder et al., 1991). Evidence has been presented that mammalian P,AR functionally couple to insect adenylyl cyclase in cells of Spodoptera frugiperda (Sf9) . However, little is known about the signal-transduction pathway in these invertebrate cells. Despite the several advantages which have made the baculovirus expression system a popular method of engineering richer sources of recombinant protein, analysis of insect proteins that are known to functionally interact with the foreign proteins has been hampered. This is because the transient expression that occurs after the cells have been infected with the recombi- nant baculovirus is a cytotoxic event that stops the transcription and translation of insect proteins and ultimately kills the cells. To facilitate further biochemical and biophysical characterization of P,AR, taking advantage of the stability and robust nature of insect ovarian cells, we have used the promoter of the IE1 gene (an immediate early gene of the baculovirus Aut...

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.

We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.