Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma.
A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.
Zhao
X X
XX
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Li
B L
BL
Langer
J A
JA
Van Riper
G
G
Pestka
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D007370:Interferon Type I; D007371:Interferon-gamma; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
D007370:Interferon Type I; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma