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 g..