Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material.
Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure-function research. Following various cloning strategies, a KM71H (Mut(s)) strain was selected. A significant concentration (74 microg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7-8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some "collagen-like" material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The "collagen-like" material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat "collagen-like" nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.
Beaulieu
Lucie
L
Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montreal, Que., Canada.
Groleau
Denis
D
Miguez
Carlos B
CB
Jetté
Jean-François
JF
Aomari
Hafida
H
Subirade
Muriel
M
D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D003094:Collagen / Q000737:chemistry; D017364:Collagenases / Q000737:chemistry; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D000070017:Pediocins; D010376:Pediococcus / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D000070017:Pediocins; D011994:Recombinant Proteins; C074288:pediocin PA-1; D003094:Collagen; D017364:Collagenases