Protein expression in yeast; comparison of two expression strategies regarding protein maturation

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50

“…These results are in striking contrast to observations for P. pastoris X-33B (producing only EntL50B), whose maximum extracellular antimicrobial activity was found in BMMY, corresponding to a bacteriocin peptide concentration 2.6-fold higher than that in BMM. The nature that caused differential peptide concentrations with the different medium compositions and different levels of bacteriocin peptide production by the bacteriocinogenic recombinant yeasts might be complex but could possibly be ascribed to one or more of the following factors: (i) (higher) aggregation of bacteriocin peptides to form oligomers and/or complexes with medium constituents that results in reduced antigen epitope recognition and, thus, also reduced antimicrobial activity (4,12,23,33,44,45,47); (ii) higher C-terminal proteolytic degradation due a high concentration of vacuolar proteases resulting from higher cell density and lysis (4,12,23); and (iii) higher recombinant gene expression due to multiple integration events (1,6,33). With regard to the latter, the higher Zeo resistance of P. pastoris X-33B than that of P. pastoris X-33A favors the possibility that the higher bacteriocin peptide concentration found in supernatants from P. pastoris X-33B may be ascribed to a higher-level multiple-integration event of entL50B, in effect resulting in a higher recombinant bacteriocin gene expression level.…”

“…Furthermore, eukaryotic genetic tools developed for gene expression in yeasts have been shown to be adaptable to bacteriocin genes (3,25,26). Despite the high versatility and efficiency of Saccharomyces cerevisiae and Pichia pastoris for the large-scale heterologous production of a variety of functional proteins (4,6,27,33,44,45), the heterologous production of enterocins by these yeast hosts has been addressed in only a few works. With regard to this, the heterologous production of biologically active enterocin L50 (L50A and L50B) by S. cerevisiae and enterocin P and hiracin JM79 by P. pastoris has demonstrated the possibility of developing bacteriocinproducing yeast strains (3,23,42).…”

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50

“…Strikingly, EntL50A and EntL50B produced in SCGR broth were more active than similar peptide amounts produced in YPGR broth. The lower antimicrobial activity found in the complex medium YPGR broth may be ascribed to one or more of the following factors: (i) the presence of compounds (e.g., salt, proteins, and/or collagen-like materials) interfering with bacteriocin activity (2,23,26,44,45), (ii) (higher) aggregation of bacteriocin peptide monomers, rendering less active oligomers and/or complexes with medium constituents (14), and/or (iii) higher proteolytic bacteriocin peptide degradation due to a higher cell density and lysis, yielding a higher concentration of vacuolar proteases (2,14,23).…”

“…At first glance, these differences may be ascribed to a higher C-terminal proteolytic degradation of EntL50A and/or to a higher secretion of EntL50B. With respect to the latter possibility, it has been demonstrated that the efficiency of secretion partially depends on the nature of the heterologous protein (6,45); however, we consider it unlikely that this phenomenon is responsible for the differences in EntL50A and EntL50B concentrations found in the respective supernatants, since these peptides show similar physicochem-FIG. 2.…”

Protein bioengineering

“…In joint research projects he has developed a multi protein expression/purification system for expression of unknown gene products and subsequent purification in a high throughput mode [11][12][13][14][15][16][17]. This work was also the basis for ongoing research projects to generate secretory protein/peptide libraries for blocking antibodies, developed during autoimmune disease [18,19].…”