Isolation of ERp72 from guinea pig term placentae using heparin Sepharose affinity chromatography.
The mammalian placenta synthesises many varied antigens, including proteins, such as hormones, enzymes and protease inhibitors. In this report, we isolated and purified the two protein isomerase-related protein precursor ERp72 isoforms from aqueous extracts of guinea pig placenta, by four (4) chromatographic procedures; i) affinity chromatography on immobilised heparin, ii) gel filtration (Ultrogel AcA-54), iii) anion exchange chromatography (Mono-Q), and, iv) negative immunoaffinity chromatography. From 20 term placentae, the final yield of ERp72 isoforms was 2.4mg (Mr 71.5 kDa) and 1.5mg (Mr 75.8 kDa). Identity was confirmed by NH2-terminal amino acid sequencing which demonstrated 85% homology to human ERp72. By indirect immunofluorecence. ER p72 expression was demonstrated in tunicamycin stressed pre-implantation embryos and unfertilised oocytes. These findings demonstrate the potential for immunological monitoring of ERp72 expression, by cultured oocytes and embryos, during manipulation by assisted reproductive technologies.
Bonifacio
M D
MD
Royal North Shore Hospital, St Leonards, New South Wales, Australia.
Steeves
T
T
Saunders
D M
DM
Sinosich
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006493:Heparin; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D012685:Sepharose; D017386:Sequence Homology, Amino Acid
D008562:Membrane Glycoproteins; C062477:endoplasmic reticulum glycoprotein p72; D006493:Heparin; D012685:Sepharose