RESUMEN
The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.
Asunto(s)
Glutatión Transferasa/genética , Receptores de Interferón/genética , Antivirales/farmacología , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular , Escherichia coli , Glutatión Transferasa/biosíntesis , Humanos , Interferón Tipo I/farmacología , Interferón-alfa , Pliegue de Proteína , Receptor de Interferón alfa y beta , Receptores de Interferón/biosíntesis , Receptores de Interferón/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes , SolubilidadRESUMEN
Some patients treated with interferon (IFN) have developed antibodies (ABs) to that IFN. We examined the incidence of such ABs in hairy cell leukemia (HCL) patients treated with IFN-alpha 2a and IFN-alpha 2b. In the initial enzyme-linked immunosorbent assay (ELISA) assays, the serum samples were tested against their corresponding IFNs. Of 73 evaluable patients treated with IFN-alpha 2b, 6 patients, who tested negative prior to treatment, were positive by ELISA following treatment with IFN. Two of the samples tested positive in the neutralization assay. Regarding the samples from patients treated with IFN-alpha 2a, 2 of the 44 patients were ELISA positive following IFN treatment (while being negative prior to treatment) and 2 others became positive in the neutralization assay following IFN treatment. Five serum samples with neutralizing activity were tested for their ability to bind to each of eight natural IFN-alpha species derived from human lymphoblastoid cells induced with Sendai virus; a different cross-reactivity profile was seen for each sample. In conclusion, a low incidence of neutralizing and nonneutralizing ABs against IFN-alpha 2 was observed in patients with HCL treated with recombinant DNA-derived IFN-alpha 2a or IFN-alpha 2b. Finally, ABs against these IFNs cross-react by ELISA with several naturally occurring IFN-alpha s.
Asunto(s)
Anticuerpos/inmunología , Interferón Tipo I/inmunología , Interferón-alfa/inmunología , Leucemia de Células Pilosas/inmunología , Anticuerpos/análisis , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Leucemia de Células Pilosas/sangre , Leucemia de Células Pilosas/terapia , Proteínas RecombinantesRESUMEN
Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.