Binding Characteristics of IFN-alpha Subvariants to IFNAR2-EC and Influence of the 6-Histidine Tag.
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.
Schmeisser
Hana
H
NIH, NIAID, Bethesda, MD 20892, USA. hschmeisser@niaid.nih.gov
Kontsek
Peter
P
Esposito
Dominic
D
Gillette
William
W
Schreiber
Gideon
G
Zoon
Kathryn C
KC
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
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D016056:Immunodominant Epitopes; D016898:Interferon-alpha; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta; D006639:Histidine