Atypical porcine type I interferon. Biochemical and biological characterization of the recombinant protein expressed in insect cells.
A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.
Niu
P D
PD
Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Lefevre
F
F
Mege
D
D
La Bonnardiere
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D007370:Interferon Type I / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D018411:Spodoptera; D013552:Swine
D006023:Glycoproteins; D007370:Interferon Type I; D011994:Recombinant Proteins