Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A.
A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy.
Andersons
D
D
Department of Microbiology, University of Stockholm, Sweden.
Engström
A
A
Josephson
S
S
Hansson
L
L
Steiner
H
H
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D015152:Blotting, Northern; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005822:Genetic Vectors; D006458:Hemolymph / Q000502:physiology; D007301:Insect Hormones / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007313:Insecta; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D010446:Peptide Fragments / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection
D023181:Antimicrobial Cationic Peptides; D007301:Insect Hormones; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D012313:RNA; C031161:cecropin A