RESUMEN
We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.
Asunto(s)
Productos del Gen gag/metabolismo , Antígenos VIH/metabolismo , Interferón gamma/metabolismo , Proteínas Virales , Animales , Anticuerpos Monoclonales/inmunología , Unión Competitiva/inmunología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Células HeLa , Humanos , Interferón gamma/farmacología , Cinética , Conejos , Proteínas Recombinantes , Sensibilidad y Especificidad , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
LamB is a membrane protein that allows the exposition of a foreign peptide on the surface of a recombinant E. coli cells. An immunopurified hybrid LamB protein has been used to elicit high-titre antibodies to a foreign epitope. Looking for a simpler purification procedure we have compared the traditional approach, which includes affinity chromatography, to continuous elution electrophoresis, in the purification of two different hybrid LamB proteins as foreign epitopes. The results obtained showed that both methods yielded the same purification, although the electrophoretic procedure had a higher yield. Continuous-elution electrophoresis could be a useful tool for the purification of membrane proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/química , Membrana Celular/ultraestructura , Cromatografía de Afinidad/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli , Modelos Estructurales , Peso Molecular , Plásmidos , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
The induction of HLA-DR antigen expression on U937 cells by interferon-gamma (IFN-gamma) is positively influenced by amount of fetal calf serum (FCS) added to the tissue culture medium. A transient alkalinization of FCS before its addition to the medium, dramatically decreased the immunomodulating activity of IFN-gamma. FCS was also found to be a dose-dependent enhancer of the IFN-gamma-induced 2',5'-oligoadenylate (2-5A) synthetase production. Our findings suggest the need for a serum factor(s), labile at basic pH values, to support at least two of the multiple IFN-gamma activities.
