Recombinant ovlFN-т Production
From 50 to 100 mg of enriched p3 or рЗТтб ovIFN-t were generated per liter of fermentation medium. The apparent molecular mass of p3 (19-20 kDa) was similar to that of the ovIFN-ts produced by ovine conceptuses. As expected, the рЗТгпб protein, which lacked the hexapeptide sequence (GDLNSP) at its carboxyl terminus, had a slightly higher electrophoretic mobility than the standard-length protein (Fig. 1A). Some protein contaminants were detected in the protein preparations; two of them probably represented cleaved GSH-transferase (—25 kDa) and uncleaved GSHtransferase/ovIFN-т fusion protein (—44 kDa). Both ovIFN-ts cross-reacted with antiserum generated from conceptus-de-rived ovIFN-т proteins (Fig. IB). buy prednisone
Effect of Carboxyl Terminus Truncation on Antiviral and Antiproliferative Activities of ovIFN-t
The p3 and рЗТгпб proteins showed similar ability to protect MDBK cells from lysis following vesicular stomatitis virus infection (Table 1). Both proteins had higher (p < 0.05) antiviral activity on bovine cells than did the boIFN-Tl used as the standard in the assay.
FIG. 1. Electrophoretic analysis of ovlFN-т (p3) and its truncated counterpart (рЗТгпб) in 12.5% SDS-PACE gels. A) Silver-stained gel containing 100 ng of each ovlFN-т. Also included were 100 ng BSA and 1 n,g of E. coli cell lysate protein, which served as controls for Western blotting. B) Western blot analysis of proteins from A that formed immunocomplexes with antiserum generated from embryo-secreted ovIFN-ts.
|TABLE 1.||Antiviral and antiproliferative activities of ovIFN-t.|
|Antiviral activity*||Antiproliferative activity*|
|p3||2.12 ± 0.92“||13.99 ± 0.85-|
|рЗТгпб||1.96 ± 0.58a||7.36 ± 0.46b|
* Antiviral activity was determined as the ability of proteins to protect MDBK cells from virus-induced cell-lysis, as compared with the activity of a laboratory standard (bolFN-Tl;5.4 X 107 lU/mg).
♦Antiproliferative activity was determined as the protein concentration required to induce a 50% inhibition in human Daudi cell number over a 4-day growth period.
a–b Results represent least-squares means and SEM from 3-4 separate studies; different superscripts within columns represent differences (p < 0.05).