Pichia Pastoris : Protein Production Host and Model Organism for Biomedical Research

Abstract

Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development. However, detailed understanding of the yeast's physiology, genetics and regulation has only developed rapidly in the last few years since published genome sequences have become available. An expanding toolbox of genetic elements and strains for the improvement of protein production is being generated, including promoters, gene copy-number enhancement, gene knockout and high-throughput methods. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. At the same time, it has become obvious that P. pastoris, as an evolutionarily more 'ancient' yeast, may in some cases be a better model for human cell biology and disease than Saccharomyces cerevisiae.

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

“…Although yeast cells typically contain about 150 proteins carrying a signal peptide, only a low number (fewer than 50 proteins) are actually secreted to the cell supernatant (Mattanovich et al, 2009). Even though P. pastoris is a very efficient host for a diverse range of heterologous proteins, there is a deficiency in useful secretion leaders (Damasceno et al, 2012;Gasser et al, 2013).…”

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting

“…We began by testing the α-factor secretion signal, consisting of the signal sequence plus pro region, because wild-type and mutant versions of this signal are commonly used to secrete foreign proteins [1,2,38,39]. Inclusion of the α-factor pro region was reportedly needed for high-level secretion of human insulin-like growth factor 1 (IGF1) from S. cerevisiae [40] and of human lysozyme from P. pastoris [9].…”

“…Budding yeasts such as Saccharomyces cerevisiae and Pichia pastoris are widely used as hosts to produce foreign proteins for research and therapeutic purposes [1][2][3][4][5]. Many of these foreign proteins traverse the secretory pathway [6,7].…”

“…A number of signal sequences have been used to secrete foreign proteins from yeasts, but the most popular is the secretion signal from S. cerevisiae pre-pro-α-factor, which is the precursor to a peptide mating pheromone [1,8,9]. Pre-pro-α-factor contains a 19-residue signal sequence that terminates in a signal peptidase cleavage site.…”

“…Finally, downstream of the dibasic Kex2 cleavage signal is an EAEA tetrapeptide, which is trimmed by the dipeptidyl aminopeptidase Ste13 [11]. Efficient secretion of foreign proteins requires the entire pre-pro-α-factor secretion signal, with or without the downstream EAEA tetrapeptide, but the secreted products are often heterogeneous due to incomplete processing by Kex2 or Ste13 [1,5].…”