Extracellular production system of heterologous peptide driven by a secretory protease inhibitor of Streptomyces.
The value of a heterologous peptide extracellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S. lividans 66 harbouring a plasmid vector constructed for SSI secretion, into which the synthetic DNA was introduced. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and amino acid analysis of the purified SSI-AP1 provided reasonable results of molecular size and composition value. Interestingly, SSI-AP1 protein showed bifunctional activity: inhibitory activity of SSI and antibacterial activity of AP1. The inhibitory activity against Escherichia coli could be also detected after the fusion protein was cleaved by factor Xa. The extracellular production system presented here should provide a useful tool for production, analysis of mode of action, and also for genetic improvement of antimicrobial peptides such as apidaecin.
Taguchi
S
S
Department of Biological Science and Technology, Science University of Tokyo, Japan.
Maeno
M
M
Momose
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001516:Bees / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics; D011480:Protease Inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C018150:subtilisin inhibitor protein, Streptomyces; C061361:apidaecin