Isolierung eines 97 kd Proteins aus dem Eileitersekret des Schweines mittels eines 'High-performance Electrophoresis-chromatography (HPEC)' Systems

Contents Isolation of a 97 kd porcine oviductal secretory protein using a high-performance electrophoresis-chromatography (HPEC) system The role of the oviductal secretory proteins at early stages of embryo development has yet to be elucidated and requires the biochemical characterization and isolation of these proteins. Because oviductal explant culture medium contains only very low protein concentrations, the aim of this study was to analyse and characterize cyclic specific proteins in a high protein content consisting of oviductal secretions, collected by can-nulation during the time of embryonic genome activation, and to isolate these proteins by means of simple purification steps in order to obtain the proteins for biological functional studies. Oviducts of gilts were cannulated and the secretions were collected at 24-h intervals on days 1–8 of the cycle. The protein pattern was analysed by SDS-polyacrylamide-gel electro-phoresis (Tris/tricine buffer system) and isoelectric focusing (native slab gels). Cyclic specific proteins were isolated using the ‘high-performance electrophoresis-chromatography’ system on 5% tube gels (Tris/tricine buffer system). The protein concentration of oviductal fluid increased from day 1 to 8 (1047.5 ± 252.6; 3992.5 ± 1097.3 mg/100 ml). A specific 97 kd protein was detected on days 1–3 (8.6, 6.8% of total proteins; laser densitometry), which was present at a lower level on the following days, and had reduced (1.9%) or not present by day 7–8. Its isoelectric point was 8 (2 D electrophoresis). The isolation of 97 kd protein from total oviductal secretion was possible within 4 h using preparative electrophoresis. Results indicated that, in spite of similarities to serum proteins, the cannulated oviductal fluid contains cyclic specific proteins. The amino-acid-sequence analysis of 97 kd protein should indicate whether it is the same protein detected in oviductal explant-conditioned medium. Using pre- parative electrophoresis, it was possible to isolate a specific protein from a total protein complex in very short time and this method is suitable for biological and chemical studies.