Generation and purification of recombinant bovine pregnancy associated glycoprotein.
Bovine pregnancy-associated glycoprotein-1 (bPAG-1) is predicted to play an essential role during pregnancy and is labelled as a potential biochemical marker of pregnancy in ungulates. We have compared the generation of the glycosylated form of recombinant bPAG-1 (rbPAG-1) by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells in attached cultures and evaluated the adaptation of the rbPAG-1 transfected cell line to suspension culture. The PAG cDNA was cloned from placental RNA obtained from a slaughtered cow on day 55 of pregnancy. The PAG-pRcRSV expression vector was transfected into HEK 293 and CHO cells. Western blot analysis showed that clonal HEK 293 cells expressed rbPAG-1 better than CHO cells in attached cultures. Transfected HEK 293 cells were adapted to suspension culture in spinner flasks and the rbPAG-1 purified to homogeneity using ion-exchange, pepstatin-sepharose affinity chromatographies and preparative SDS-PAGE. The expression of rbPAG-1 was immunocharacterised using a polyclonal antibody. Our findings indicated that 293 cells are suitable for production of glycosylated form of rbPAG-1 and that the availability of the recombinant glycoprotein will aid in further studies to elucidate the function and structure of the protein.
Patel
O V
OV
Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Takahashi
T
T
Imai
K
K
Hashizume
K
K
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D002417:Cattle; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary / Q000032:analysis*; D005260:Female; D006801:Humans; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection / Q000662:veterinary
D018076:DNA, Complementary; D011257:Pregnancy Proteins; D011994:Recombinant Proteins