Abstract: 544

PRODUCTION, PURIFICATION, AND C-TERMINAL SEQUENCING OF BIOACTIVE RECOMBINANT BOVINE ISG17.

JK Pru¹ , KJ Austin^{1 *}, TR Hansen^{1 *}
Department of Animal Science, University of Wyoming, Laramie, Wyoming ¹

A bovine (bo) interferon (IFN) stimulated gene encodes a 17-kDa ubiquitin homolog called ISG17 that covalently links to cytosolic endometrial proteins and is released into the uterine lumen in response to pregnancy. The objective of this experiment was to generate bioactive recombinant (r) boISG17 using the methylotrophic yeast P. pastoris. The ISG17 coding region was inserted into a pPICZaA expression vector behind the a-factor signal sequence to promote secretion of rboISG17. Positive transformants were selected on YPD-zeocin plates and gene insertion was confirmed by PCR. Transformants containing single insertions expressed protein at 5-7 mg/liter. Several multi-gene insertion transformants were identified via Southern blot analysis that produced rboISG17 in concentrations > 25 mg/liter. The greatest expression of rboISG17 occurred in non-buffered MMY media at 48 h with an inoculation OD₆₀₀ of 0.4. Carboxy-terminal sequencing of purified (> 95%; ion exchange/gel filtration) rboISG17 revealed retention of C-terminal Gly-Gly residues that have been shown to be hydrolyzed from r human (hu) ISG15 using a bacterial expression system because of carboxypeptidase activity. Retention of these residues is a critical functional requirement in the covalent ligation of ubiquitin and huISG15 to targeted proteins. Purified rboISG17 (50 and 500 ng/ml) induced expression of boIFN-gamma by peripheral blood mononuclear cells (PBMC) as determined by RT-PCR amplification of a 376 bp cDNA. This cDNA was identical in size to one amplified from PBMCs treated with Con A (10 ng/ml) and another from plasmid containing the boIFN-gamma cDNA. It is concluded that rboISG17, generated in a yeast expression system, retains C-terminal Gly-Gly residues and extracellular bioactivity. NIH HD 32475.

    This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.