Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus.
From a cDNA library made from fatbody of immunized Hyalophora cecropia pupae, a full-length clone corresponding to basic attacin was isolated. The corresponding amino acid sequence suggests that basic attacin is synthesized as a 233-residue preproprotein. This was confirmed by cloning the cDNA fragment encoding the basic attacin in the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter and expressing the protein in Spodoptera frugiperda (Sf9) cells. Biologically active attacin was also produced in last instar larvae of Trichoplusia ni after injection of recombinant virus. The concentration obtained was 300-500 times that obtained in cell culture supernatants. In cell culture the induction kinetics of attacin were followed at both transcriptional and translational levels. From the protein processing pattern it was suggested that a protease produced by the Spodoptera cells cleaves attacin at the double arginine residues at positions 45 and 46. The instability of the attacin proteins is rationalized in terms of their random-coil structure which was deduced from circular dichroism measurements.
Gunne
H
H
Department of Microbiology, University of Stockholm, Sweden.
Hellers
M
M
Steiner
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002942:Circular Dichroism; D004247:DNA / Q000032:analysis; D005796:Genes; D005814:Genes, Viral; D015151:Immunoblotting; D007301:Insect Hormones / Q000096:biosynthesis*; D019476:Insect Proteins; D007304:Insect Viruses / Q000235:genetics*; D007915:Lepidoptera / Q000378:metabolism*; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism*; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
D007301:Insect Hormones; D019476:Insect Proteins; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C039711:attacin antibacterial protein, insect; D004247:DNA