title: Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha.
abstract: A phosphorylation site was introduced into human interferon alpha A (IFN-alpha A) by site-specific mutation of the coding sequence. Three slightly different phosphorylation sites were created by using the predicted amino acid consensus sequences for phosphorylation by the cAMP-dependent protein kinase. The resultant modified interferons (IFN-alpha A-P) were expressed in Escherichia coli and purified. The purified proteins exhibit antiviral activity on bovine and human cells similar to that of the unmodified IFN-alpha A. The IFN-alpha A-P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity (2000-5000 Ci/mmol; 1 Ci = 37 GBq) with retention of biological activity. The 32P-labeled IFN-alpha A-P proteins bind to cells and can be covalently bound to the IFN-alpha/beta receptor with a bifunctional reagent as can human IFN-alpha A. The introduction of phosphorylation sites into proteins provides a procedure to prepare a large variety of radioactive proteins for research and clinical use.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
authors.forename: B L
authors.initials: BL
authors.lastname: Li
authors.forename: J A
authors.initials: JA
authors.lastname: Langer
authors.forename: B
authors.initials: B
authors.lastname: Schwartz
authors.forename: S
authors.initials: S
authors.lastname: Pestka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids
chemical_list: D007370:Interferon Type I; D004247:DNA