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.
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