Purification of bovine trophoblast protein-1 complex and quantification of its microheterogeneous variants as affected by culture conditions.
The bovine trophoblast protein-1 complex (bTP-1) is a group of glycosylated interferon-alpha 11, molecules secreted by the bovine conceptus that plays a critical role in preventing luteolysis during early pregnancy. In the current studies, secretion of individual variant forms of bTP-1 was quantified under a variety of culture conditions that could affect yields of bTP-1 for preparative-scale production of bTP-1. Additionally, a purification scheme for bTP-1 was developed. Conceptuses from Day 17 produced 13 proteins in the molecular weight and pI range characteristic of bTP-1, with 4-5 isoforms (pI = 5.6-6.6) at each of three molecular weight classes (21, 23.2 and 25.8 kDa). The major forms of bTP-1 were two variants of 23.2 kDa having pIs of 6.2 and 6.6. The relative proportion of bTP-1 variants was generally unaffected by culture conditions. Cultured conceptuses secreted bTP-1 at a sustained rate for 3 days and gaseous environment was without major effect on bTP-1 secretion. Conceptuses from superovulated cows also produced bTP-1 at Day 17 of pregnancy, suggesting that superovulation might be a useful method for increasing total bTP-1 yield per cow. The purification scheme that was developed utilized ultrafiltration with Centricon devices to achieve rapid molecular weight fractionation, desalting and concentration of conceptus secretory proteins prior to purification of bTP-1 using anion-exchange and gel filtration HPLC. The resultant preparation of bTP-1 included 9 variant forms of bTP-1 as well as a slight amount of a 45-kDa contaminant. Purified bTP-1 possessed antiviral activity but the specific activity was apparently reduced when conceptus-conditioned medium used for purification was stored for prolonged periods.
Plante
C
C
Department of Diary Science, University of Florida, Gainesville.
Hansen
P J
PJ
Thatcher
W W
WW
Johnson
J W
JW
Pollard
J W
JW
Mirando
M A
MA
Bazer
F W
FW
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D046508:Culture Techniques; D004622:Embryo, Mammalian / Q000276:immunology; D005260:Female; D007370:Interferon Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007526:Isoelectric Point; D007536:Isomerism; D008970:Molecular Weight; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D007370:Interferon Type I; D011257:Pregnancy Proteins; C020947:interferon tau