The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon).
Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Klemann
S W
SW
Department of Animal Sciences, University of Missouri, Columbia 65211.
Li
J Z
JZ
Imakawa
K
K
Cross
J C
JC
Francis
H
H
Roberts
R M
RM
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D046508:Culture Techniques; D004262:DNA Restriction Enzymes; D004355:Drug Stability; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D007370:Interferon Type I; D008969:Molecular Sequence Data; D010957:Plasmids; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D012756:Sheep
D000998:Antiviral Agents; D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau; D004262:DNA Restriction Enzymes