RESUMEN
We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.
Asunto(s)
Antígenos/genética , Tromboplastina/genética , Vacunas Sintéticas , Vacunas , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Secuencia de Bases , Carbohidratos/análisis , Células Cultivadas , Expresión Génica , Glicosilación , Humanos , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Simplexvirus/genética , Simplexvirus/inmunología , Tromboplastina/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Recombinant human transforming growth factor alpha (TGF alpha), which is active as assessed by competition with epidermal growth factor (EGF) for binding to the EGF receptor, has been produced in Escherichia coli and separated from misfolded and inactive forms of recombinant TGF alpha using reverse-phase high performance liquid chromatography. The purified recombinant TGF alpha was used to produce a monoclonal antibody that binds to active TGF alpha specifically. The antibody was coupled to Sepharose and used as an independent method for purifying active TGF alpha. The EGF receptor binding activity of antibody affinity purified TGF alpha is comparable to that of high performance liquid chromatography-purified active TGF alpha, and is 0.55 mg of EGF eq/mg of TGF alpha. The disulfide arrangement of the active TGF alpha was determined after digestion with thermolysin, and found to be analogous to the disulfide arrangement previously determined for EGF (Savage, C. R., Hash, J. H., and Cohen, S. (1973) J. Biol. Chem. 248, 7666-7672).