Characterization of immunorelated peptides to porcidin P1.
Porcidin P1, an antimicrobial peptide purified from the granules of porcine polymorphonuclear neutrophils (PMN) using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC), was covalently conjugated to BSA and used to generate monospecific polyclonal ascites. Antibodies raised against porcidin P1 were covalently coupled to an Affi-gel Hz affinity column and used for immunoaffinity chromatography of peptides from porcine PMN cell extract. Eleven immunorelated peptides were eluted from the column from neutrophil cell extracts and purified to homogeneity by HPLC. The molecular weights of the immunorelated peptides were determined by mass spectral analysis and ranged in size from 1.91 to 10.65 kDa. Of the 11 immunorelated peptides which were bound to the affinity column, only six peptides were recognized by the anti-porcidin antibodies after HPLC purification. Three immunoreactive peptides displayed potent antibacterial activity towards Staphylococcus aureus and Escherichia coli, reducing viability by as much as 99.9% (> 3 log reduction in CFU) when 5 mu g/mL of each purified peptide was used. The polyclonal monospecific antibodies also reacted with proteins from ovine and human PMN, illustrating possible structural relationships between small antibacterial peptides from the different species.
Alberdi
F
F
Department of Applied Biology and Biotechnology, Royal Melbourne Institute of Technology, Victoria, Australia.
Alderton
M R
MR
Coloe
P J
PJ
Smith
S C
SC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000737:chemistry* / Q000276:immunology; D012756:Sheep; D013552:Swine
D000596:Amino Acids; D001798:Blood Proteins