Generation and Characterization of Recombinant Unmodified and Phosphorylatable Murine IFN- a1 in the Methylotropic Yeast Pichia pastoris

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFNa1 (rMuIFN-a1) protein was expressed in the methylotropic yeast Pichia pastoris . rMuIFN-a1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN- a1P) to enable radiolabeling by g 32 P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN- a1) and 25.5 (MuIFN- a1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN- a1 proteins produced in P. pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation. The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing. The MuIFN- a1 and MuIFN- a1P protein preparations had specific antiviral activities of 1.3 3 10 7 and 4.7 3 10 6 IU/mg protein, respectively. MuIFN- a1P could be radiolabeled to a high specific radioactivity (0.6‐ 2 3 10 8 cpm/mg protein) with g 32 P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties. Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of g 32 P-ATP-MuIFN- a1P to cell surface type I IFN receptors.