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Isolation of an ovine pregnancy specific protein
THERIOGENOLOGY
ISOLATION OF AN OVINE PREGNANCY SPECIFIC PROTEIN ZOLI, A.P.; BECKERS, J.F.; ECIORS, F. Dept. of Animal Endocrinology and Reproduction (IRSIA Research Unit) Faculty of Veterinary Medicine (ULg) Rue des vCt&inaires,45 B-1070 Brussels Belgium Bovine Pregnancy Specific proteins (bPSP) were isolated and partially purified from bovine fetal membranes (Butler et al., BiolReprod.,l982& 925). One of these is being used for early pregnancy diagnosis in cattle (Sasser et al., Biol.Reprod., 1987, s 936). In spite of a large immunologic homology of these proteins in different ruminant species, Ruder et al. (1988, Therio, a, 905) showed that the inhibition curve of pregnant ovine serum in serial dilutions was not parallel to the bovine standard curve. This observation was confirmed in our laboratory using a radioimmunoassay (RIA) developed earlier for a bovine pregnancy specific protein (Beckers et al.,Therio, 1988, a, 219). The lack of identity between ovine and bovine PSP justified isolation of the ovine pregnancy proteins. Uteri were collected from pregnant ewes at slaughter, the fetal cotyledons were immediately dissected and stored at -20°C. Samples were thawed, minced and homogeneized in kalium phosphate buffer (.Ol M; pH 7.6) and the homogenate stirred slowly at 4’C for 16 hours. The homogenate was then centrifuged at 27,000 g for one hour at 4’C. The proteins in supematant were precipitated using ammonium sulfate (A.S). The 40-80 % A.S. precipitate was dialysed against Tris-HCI buffer (01 M; pH 8) and chromatographed on DEAE-Sephadex DE-52 (Whatmann), the column was eluted in four steps of increasing ionic strength buffer (by adding NaCl). The bovine PSP RIA was used to monitor the ovine pregnancy protein in each step. The active fraction was then dialysed against kalium phosphate buffer (.Ol M; pH 5.5) and chromatographed on Sulfopropyl Sephadex C-25 (Pharmacia). The bound proteins were eluted using a linear salt gradient (0 to .3 M NaCl). Fractions of 8 ml were collected and proteins were monitored by UV absorption at 280 nm. The RTA positive fractions were pooled, concentrated by ultrafiltration to about 10 mg proteins per ml of buffer and further fractionated by gel filtration column chromatography (Sephacryl S -200 HR, Pharmacia). The positive fractions were pooled and lyophilized. The ovine PSP preparation is homogeneous in native and SDS-PAGE. Molecular weight and isoelectric point are similar to the bovine PSP (60,000 Daltons and 5.5 respectively). This work is being continued in order to develop a homologous RIA for the ovine PSP and to compare the ovine and bovine proteins both structurally and immunologically .
366
JANUARY
1990 VOL. 33 NO. 1
ISOLATION OF AN OVINE PREGNANCY SPECIFIC PROTEIN ZOLI, A.P.; BECKERS, J.F.; ECIORS, F. Dept. of Animal Endocrinology and Reproduction (IRSIA Research Unit) Faculty of Veterinary Medicine (ULg) Rue des vCt&inaires,45 B-1070 Brussels Belgium Bovine Pregnancy Specific proteins (bPSP) were isolated and partially purified from bovine fetal membranes (Butler et al., BiolReprod.,l982& 925). One of these is being used for early pregnancy diagnosis in cattle (Sasser et al., Biol.Reprod., 1987, s 936). In spite of a large immunologic homology of these proteins in different ruminant species, Ruder et al. (1988, Therio, a, 905) showed that the inhibition curve of pregnant ovine serum in serial dilutions was not parallel to the bovine standard curve. This observation was confirmed in our laboratory using a radioimmunoassay (RIA) developed earlier for a bovine pregnancy specific protein (Beckers et al.,Therio, 1988, a, 219). The lack of identity between ovine and bovine PSP justified isolation of the ovine pregnancy proteins. Uteri were collected from pregnant ewes at slaughter, the fetal cotyledons were immediately dissected and stored at -20°C. Samples were thawed, minced and homogeneized in kalium phosphate buffer (.Ol M; pH 7.6) and the homogenate stirred slowly at 4’C for 16 hours. The homogenate was then centrifuged at 27,000 g for one hour at 4’C. The proteins in supematant were precipitated using ammonium sulfate (A.S). The 40-80 % A.S. precipitate was dialysed against Tris-HCI buffer (01 M; pH 8) and chromatographed on DEAE-Sephadex DE-52 (Whatmann), the column was eluted in four steps of increasing ionic strength buffer (by adding NaCl). The bovine PSP RIA was used to monitor the ovine pregnancy protein in each step. The active fraction was then dialysed against kalium phosphate buffer (.Ol M; pH 5.5) and chromatographed on Sulfopropyl Sephadex C-25 (Pharmacia). The bound proteins were eluted using a linear salt gradient (0 to .3 M NaCl). Fractions of 8 ml were collected and proteins were monitored by UV absorption at 280 nm. The RTA positive fractions were pooled, concentrated by ultrafiltration to about 10 mg proteins per ml of buffer and further fractionated by gel filtration column chromatography (Sephacryl S -200 HR, Pharmacia). The positive fractions were pooled and lyophilized. The ovine PSP preparation is homogeneous in native and SDS-PAGE. Molecular weight and isoelectric point are similar to the bovine PSP (60,000 Daltons and 5.5 respectively). This work is being continued in order to develop a homologous RIA for the ovine PSP and to compare the ovine and bovine proteins both structurally and immunologically .
366
JANUARY
1990 VOL. 33 NO. 1