Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector.
Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.
Kato
Y
Y
Biosignal Research Center, Gunma University, Japan.
Sato
I
I
Ihara
T
T
Tomizawa
K
K
Mori
J
J
Geshi
M
M
Nagai
T
T
Okuda
K
K
Kato
T
T
Ueda
S
S
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005640:Follicle Stimulating Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006107:Granulosa Cells / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D009865:Oocytes; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013552:Swine; D014162:Transfection
D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone