Protein Expression and PuriWcation 43 (2005) 111–125 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter. Crown copyright  2005 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.05.012 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 Lucie Beaulieu a,b , Denis Groleau a,¤ , Carlos B. Miguez a , Jean-François Jetté a , HaWda Aomari a , Muriel Subirade b,¤ a Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2 b Canada Research Chair in Proteins, Biosystems and Functional Foods, Dairy Research Centre (STELA), Nutraceuticals and Functional Foods Institute (INAF), Université Laval, Que., Canada G1K 7P4 Received 7 February 2005, and in revised form 18 May 2005 Available online 22 June 2005 Abstract 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 signiWcant concentration (74 g/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant Xuids from P. pastoris cultures, har- boring 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 diVerently 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 diVerences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that ped- iocin PA-1 may possess a somewhat “collagen-like” nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into Wbrils. Crown copyright  2005 Published by Elsevier Inc. All rights reserved. Keywords: Pediocin PA-1; Pichia pastoris; Biological activity; Inhibition; Collagen-like material Strains of lactic acid bacteria (LAB) 1 produce a wide variety of bacteriocins. These antimicrobial peptides, con- taining 20–60 amino acids, display both cationic and hydrophobic properties [1]. Bacteriocins act primarily at the cytoplasmic membrane of Gram-positive bacteria, and, in many cases, on bacteria closely related to the producing strain [1,2] . They have been extensively studied but, despite these investigations, only a small number have demon- strated signiWcant economic potential for use in the food and biomedical Welds [3]. Studies on their genetic determi- nants far outweigh studies on their biochemical structure. This may be due to the diYculties of obtaining suYcient amounts of puri Wed bacteriocin [4]. Constant eVorts have been pursued to produce bacteriocins at various scales and to optimize both production and puri Wcation. * Corresponding authors. Fax: +1 514 496 7251 (D. Groleau); +1 418 656 3353 (M. Subirade). E-mail addresses: Denis.Groleau@cnrc-nrc.gc.ca (D. Groleau), Muriel.subirade@aln.ulaval.ca (M. Subirade). 1 Abbreviations used: LAB, lactic acid bacteria; LB, Luria–Bertani, LSLB, low salt Luria–Bertani; CB, coating buVer; PBS, phosphate- buVered saline; ADT, agar diVusion test; CV, column volume; PVDF, polyvinylidene Xuoride; PTH-aa, phenylthiohydantion amino acid; HIC, hydrophobic interaction chromatography.