Construction and Activity of Phosphorylatable Human Interferon-αB2 and Interferon-αA/D

Abstract

The polymerase chain reaction (PCR) was used to introduce a phosphorylation site into human interferon-αB2 (Hu-IFN-αB2) and the chimeric human interferon-αA/D (Hu-IFN-αA/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-αB2-P and Hu-IFN-αA/D-P) were expressed in Escherichia coli and purified. The purified proteins exhibited antiviral activities similar to that of unmodified Hu-IFN-αB2 and Hu-IFN-αA/D. The Hu-IFN-αB2-P and Hu-IFN-αA/D-P can be phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and [γ-³²P]ATP with retention of biological activities. The introduction of phosphorylation sites into Hu-IFN-αB2 and Hu-IFN-αA/D provides new reagents for studies of receptor binding, pharmacokinetics, and other studies where labeled IFNs are useful.