Generation and Characterization of Recombinant Unmodified and Phosphorylatable Murine IFN-α1 in the Methylotropic Yeast Pichia pastoris

Abstract

In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-α1 (rMuIFN-α1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-α1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-α1P) to enable radiolabeling by γ³²P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-α1) and 25.5 (MuIFN-α1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-α1 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-α1 and MuIFN-α1P protein preparations had specific antiviral activities of 1.3 × 10⁷ and 4.7 × 10⁶ IU/mg protein, respectively. MuIFN-α1P could be radiolabeled to a high specific radioactivity (0.6-2 × 10⁸ cpm/μg protein) with γ³²P-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 γ³²P-ATP-MuIFN-α1P to cell surface type I IFN receptors.