title: Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris.
abstract: In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris. rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated. Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies. The MuIFN-alpha1 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-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively. MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(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 gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors.
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D016898:Interferon-alpha / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D016898:Interferon-alpha; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases