Homogeneous production of feline interferon in silkworm by replacing single amino acid code in signal peptide region in recombinant baculovirus and characterization of the product.
In a previous paper we reported that we constructed a recombinant baculovirus named BmFeIFN1 which produced recombinant feline interferon (FeIFN) in silkworm after infection. High purification of FeIFN in body fluid from the larvae revealed that two kinds of FeIFN were produced by the BmFeIFN1. The difference between the two in the NH2-terminal amino acid sequence was clarified, and two kinds of processing at different sites in FeIFN precursor were suggested. Changing one residue of the amino acid sequence in deduced signal peptide by site directed mutagenesis of encoding cDNA led to the production of homogeneous FeIFN whose NH2-terminal three amino acid sequence was identical to the conserved sequence of human interferon-alpha. We designated the FeIFN produced as rFeIFN. Amino acid sequence analysis revealed that rFeIFN consisted of 170 amino acid residues. The COOH-terminal amino acid of the rFeIFN is Glu which is located at one amino acid residue upstream to the COOH-terminal amino acid deduced from the nucleotide sequence. Positive periodic acid Schiff reaction, undetectability of Asn at position 79 by automated protein sequencer, and detectability of N-acetylglucosamine not N-acetylgalactosamine suggested that rFeIFN was N-glycosylated. rFeIFN was stable at pH 1.5 at 4 degrees C for at least 50 days, and had an antiviral action on feline calicivirus or feline herpesvirus in vitro. Its isoelectric point was pI 6.5. Comparing the structure and biosynthesis of rFeIFN with those of HuIFNs, rFeIFN may be classified as omega-type.
Ueda
Y
Y
Basic Research Laboratories, TORAY Industries, Inc., Kanagawa, Japan.
Sakurai
T
T
Yanai
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001681:Biological Assay; D012831:Bombyx; D002415:Cats; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006801:Humans; D007372:Interferons / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D011995:Recombination, Genetic; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004247:DNA; D007372:Interferons