Rational redesign of porcine pepsinogen containing an antimicrobial peptide.
A novel strategy for the controlled release and localization of bioactive peptides within digestive and immunity-related enzymes was developed. The N-terminus of porcine pepsinogen A was fused to the basic amino acid-rich region of bovine lactoferricin B termed 'tLfcB', a cationic antimicrobial/anticancer peptide. Recombinant tLfcB-porcine pepsinogen A was expressed in soluble form in Escherichia coli as a thioredoxin (Trx) fusion protein. Thioredoxin-tLfcB-porcine pepsinogen A was found to activate autocatalytically under acidic conditions. Recombinant pepsin A derived from the activation of the fusion protein had a catalytic rate and substrate affinity similar to that derived from the recombinant thioredoxin-porcine pepsinogen A control. Pepsin-treated thioredoxin-tLfcB-porcine pepsinogen A yielded increased antimicrobial activity against the Gram-negative bacteria E.coli relative to control suggesting that a second function (antimicrobial activity) was successfully engineered into a functional peptidase. The novel design strategy described herein presents a potential strategy for targeted delivery of antimicrobial or therapeutic peptides in transgenic organisms via re-engineering native proteins critical to plant and animal defense mechanisms.
Bryksa
Brian C
BC
Department of Food Science, University of Guelph, Guelph, ON, Canada N1G 2W1.
Horimoto
Yasumi
Y
Yada
Rickey Y
RY
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D020116:Pepsinogen A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013552:Swine; D053719:Tandem Mass Spectrometry; D013879:Thioredoxins / Q000235:genetics
D023181:Antimicrobial Cationic Peptides; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; C082171:lactoferricin B; D013879:Thioredoxins; D020116:Pepsinogen A; D007781:Lactoferrin