title: Binding Characteristics of IFN-alpha Subvariants to IFNAR2-EC and Influence of the 6-Histidine Tag.
abstract: The expression, purification, detection, and assay of recombinant proteins have been made more convenient and rapid by the use of small affinity tags. To facilitate the purification of interferon-alpha2c (IFN-alpha2c) by metal chelate affinity chromatography, N-terminal 6-histidine tag was introduced via genetic manipulation. Two preparations of IFN material were purified; one contained IFN-alpha2c with the 6-histidine tag, and the other contained IFN-alpha2c without the 6-histidine tag. The antigenic properties of the human IFN-alpha2c subvariant with and without the 6-histidine tag, as well as the effects of the N-terminal 6-histidine tag on IFN-alpha2c interaction with the extracellular domain of human IFN-alpha receptor chain 2 (IFNAR2-EC) were examined. For the purposes of this study, IFNs were characterized by Western blots with anti-IFN monoclonal antibodies (mAb) and bioassays. Immunoblot analyses showed differences between IFN-alpha2c-6-histidine tag and IFN-alpha2a, b, c in their interaction with IFNAR2-EC. We also observed differences between IFN-alpha2c-6-histidine tag and IFN-alpha2a, b, c in bioactivities. This study is the first report that shows that an N-terminal 6-histidine tag on IFN-alpha2c can affect its interaction with receptor and cause a different bioactivity.
authors.affiliation: NIH, NIAID, Bethesda, MD 20892, USA. hschmeisser@niaid.nih.gov
authors.forename: Hana
authors.initials: H
authors.lastname: Schmeisser
authors.forename: Peter
authors.initials: P
authors.lastname: Kontsek
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.forename: William
authors.initials: W
authors.lastname: Gillette
authors.forename: Gideon
authors.initials: G
authors.lastname: Schreiber
authors.forename: Kathryn C
authors.initials: KC
authors.lastname: Zoon
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000998:Antiviral Agents / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006639:Histidine / Q000235:genetics / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D053633:Receptor, Interferon alpha-beta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D016056:Immunodominant Epitopes; D016898:Interferon-alpha; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta; D006639:Histidine