Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1: purification and antiviral activity.
Ovine trophoblast protein-1 (oTP-1) is a unique, Type I, trophoblast interferon (IFN) that possesses potent antiviral activity and is thought to be primarily responsible for maternal recognition of pregnancy in sheep. To provide sufficient amounts of protein for detailed studies, a synthetic gene for oTP-1 was designed and assembled in Escherichia coli, subcloned into a yeast expression plasmid, and used to overproduce recombinant oTP-1 in Saccharomyces cerevisiae. Recombinant oTP-1 was purified from soluble yeast extract using sequential ion-exchange and molecular sieve chromatography. Recombinant oTP-1 purified in this fashion exhibited potent antiviral activity (0.6 x 10(8) U/mg) similar to native oTP-1. This expression system will enable production of large quantities of soluble, biologically active, and correctly processed recombinant oTP-1. Furthermore, the synthetic gene construct facilitates introduction of mutations for ongoing structure/function studies of this unique, Type I, trophoblast IFN.
Ott
T L
TL
Department of Animal Science, University of Florida, Gainesville 32611.
Van Heeke
G
G
Johnson
H M
HM
Bazer
F W
FW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D007370:Interferon Type I / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008186:Luteolytic Agents / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012756:Sheep
D007370:Interferon Type I; D008186:Luteolytic Agents; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau