Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma

Analytical Biochemistry
X X ZhaoS Pestka

Abstract

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 o...Continue Reading

References

Jul 1, 1985·The Journal of General Virology·B Robert-GalliotC Chany
Jan 1, 1987·Annual Review of Biochemistry·S PestkaC E Samuel
Apr 29, 1986·Biochemical and Biophysical Research Communications·T ArakawaP H Lai
Aug 1, 1988·Journal of Interferon Research·R FieldsS Pestka
Mar 1, 1985·Molecular and Cellular Biology·R A WattM Rosenberg
Mar 1, 1983·Archives of Biochemistry and Biophysics·N T ChangS Pestka

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Citations

Jun 1, 1995·Pharmacology & Therapeutics·M A Horisberger, S Di Marco
May 10, 2001·Proceedings of the National Academy of Sciences of the United States of America·C S HanR M Roberts
Sep 27, 2003·Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research·S TrajanovskaP J Hertzog

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