Recombinant expression of the antimicrobial peptide polyphemusin and its activity against the protozoan oyster pathogen Perkinsus marinus.
Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.
Pierce
J C
JC
Department of Biological Sciences, Philidelphia College of Pharmacy and Science, Pennsylvania 19104-4495, USA. j.pierce@pcps.edu
Maloy
W L
WL
Salvador
L
L
Dungan
C F
CF
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D016782:Apicomplexa / Q000187:drug effects*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006737:Horseshoe Crabs / Q000737:chemistry; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010125:Ostreidae / Q000469:parasitology*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
D018528:ATP-Binding Cassette Transporters; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C062385:polyphemusin I