Production, purification, and carboxy-terminal sequencing of bioactive recombinant bovine interferon-stimulated gene product 17

An interferon (IFN)-stimulated gene (ISG) encodes a bovine 17-kDa protein ( bISG17) that is released from endometrial cells but also conjugates to intr acellular proteins through a ubiquitin-like mechanism. During early pregnan cy in ruminants, conceptus-derived IFN-tau induces endometrial ISG17. The p resent experiments were designed to generate bioactive recombinant (r) bISG 17. The Pichia pastoris yeast expression system was used because previous e xperiments expressing the human ISG15 ortholog in bacteria were confounded by inherent carboxypeptidase activity that cleaved C-terminal residues resu lting in an inactive protein. In a series of extensive yeast culture experi ments using shaker-bath and fermentation approaches, optimal conditions wer e determined for a transformant containing a multi-ISG17 gene insertion. Re combinant bISG17 was purified. Carboxy-terminal sequencing revealed that rb ISG17 retained the C-terminal Gly that is potentially critical for the firs t step in covalent attachment to targeted intracellular proteins. The rISG1 7 induced (P < 0.0001) IFN-gamma mRNA (reverse transcription-polymerase cha in reaction) and release of IFN-gamma protein (ELISA) by bovine peripheral blood mononuclear cells. The IFN-gamma mRNA also was upregulated (P < 0.000 1) in endometrium from pregnant (Day 18) when compared with nonpregnant (Da ys 14 and 18) cows. It is concluded that rbISG17 generated in a yeast expre ssion system retains cytokine/hormonal activity This is the first descripti on coupling the biology of two distinct IFNs (gamma and tau) through the in termediary ubiquitin homolog ISG17.