RESUMO
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.
Assuntos
Fragmentos de Imunoglobulinas/imunologia , Imuno-Histoquímica/métodos , Escherichia coli/genética , Corantes Fluorescentes , Compostos de Ouro , Halobacterium salinarum/genética , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Fragmentos de Imunoglobulinas/química , Imuno-Histoquímica/métodos , Anticorpos Monoclonais/genética , Antígenos de Superfície/análise , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Fragmentos de Imunoglobulinas/genéticaRESUMO
The topology of the ubiquinol:cytochrome-c oxidoreductase (cytochrome bc1 complex) from Paracoccus denitrificans was investigated by immunoelectron microscopy with sequence-specific murine monoclonal antibodies. Epitope mapping with synthetic peptides and enzymic proteolytic cleavage of the cytochrome bc1 complex were employed to localize precisely the respective antibody epitopes on the subunits of this membrane protein complex. Localization of defined epitopes on the cytochrome bc1 complex by immunoelectron microscopy clearly demonstrates that the N-terminus of the cytochrome b subunit is exposed to the periplasmic space. This finding is in agreement with a nine-transmembrane-helices topology model (I-IX) as predicted before for cytochrome b. However, due to other published evidence we favour the existence of an additional transmembrane helix (helix 0) complementing a more recently published eight-helices model (A-C,cd, D-H), at least for prokaryotes.
Assuntos
Anticorpos Monoclonais/imunologia , Grupo dos Citocromos b/química , Complexo III da Cadeia de Transporte de Elétrons/química , Mapeamento de Epitopos , Paracoccus denitrificans/enzimologia , Sequência de Aminoácidos , Complexo III da Cadeia de Transporte de Elétrons/imunologia , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently insufficient for structural studies. To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments. cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column. The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and subcomplexes or individual subunits thereof. These membrane proteins were purified simply by combining the crude P. denitrificans membrane preparation with the E. coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidin affinity chromatography. Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag. The affinity column could thus be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.
Assuntos
Cromatografia de Afinidade/métodos , Fragmentos de Imunoglobulinas/genética , Proteínas de Membrana/isolamento & purificação , Engenharia de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Complexo III da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Hibridomas/imunologia , Técnicas Imunológicas , Indicadores e Reagentes , Dados de Sequência Molecular , Paracoccus denitrificans/química , EstreptavidinaRESUMO
Twelve-hour exposure of G1 Ehrlich ascites cells to controlled hypoxia (200 ppm of O2 at 1 bar) suppressed replicon initiation. Synchronous cycling, beginning with a normal S phase, was released by reoxygenation immediately. The addition of cycloheximide at reoxygenation largely resuppressed, after a short initial burst, succeeding replicon initiations. Alkaline sedimentation analysis of growing daughter strand DNA, DNA fiber autoradiography, and analysis of the newly formed DNA demonstrated that normal chain growth and DNA maturation (replicon termination) in the initially activated replicons continued in the presence of cycloheximide. After 2 to 3 h, a low level of cycloheximide-insensitive background replication emerged out of the then-ebbing single surge of activity of the initially released replicons.
Assuntos
Replicação do DNA , DNA/biossíntese , Replicon , Fase S , Animais , Autorradiografia , Carcinoma de Ehrlich , Cicloeximida/farmacologia , Replicação do DNA/efeitos dos fármacos , Hipóxia , Técnicas In Vitro , Camundongos , Mapeamento por Restrição , Fase S/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human beta 2-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350,000 human receptors/cell. 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 alpha subunit of stimulatory GTP-binding proteins cross-react with the insect alpha subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The beta subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the C-terminal amino acids of the mammalian beta subunit of GTP-binding proteins, but is about approximately 2 kDa larger than that of the beta 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.