Construction and activity of phosphorylatable human interferon-alpha B2 and interferon-alpha A/D.
The polymerase chain reaction (PCR) was used to introduce a phosphorylation site into human interferon-alpha B2 (Hu-IFN-alpha B2) and the chimeric human interferon-alpha A/D (Hu-IFN-alpha A/D). The phosphorylation sites were created by adding an amino acid consensus sequence for phosphorylation by the cAMP-dependent protein kinase to the carboxyl termini of the IFNs. The resultant modified IFNs (Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P) were expressed in Escherichia coli and purified. The purified proteins exhibited antiviral activities similar to that of unmodified Hu-IFN-alpha B2 and Hu-IFN-alpha A/D. The Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P can be phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and [gamma-32P]ATP with retention of biological activities. The introduction of phosphorylation sites into Hu-IFN-alpha B2 and Hu-IFN-alpha A/D provides new reagents for studies of receptor binding, pharmacokinetics, and other studies where labeled IFNs are useful.
Wang
P
P
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854-5635.
Izotova
L
L
Mariano
T M
TM
Donnelly
R J
RJ
Pestka
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D016898:Interferon-alpha; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins
D007370:Interferon Type I; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C076799:interferon-alpha 8; C080665:interferon-alpha A-D