Screening Endogenous Signal Peptides and Protein Folding Factors to Promote the Secretory Expression of Heterologous Proteins in Pichia Pastoris

Secretory expression is most often desired but usually hampered by limitations of signal peptide processing and protein folding in the methylotrophic yeast Pichia pastoris. To alleviate such limitations, novel endogenous signal peptides (Dan4, Gas1, Msb2, and Fre2) and folding factors (Mpd1p, Pdi2p, and Sil1p) were predicted based on the reported P. pastoris secretome and genome. Their effects were investigated using three reporter proteins: yeast-enhanced green fluorescent protein (yEGFP), β-galactosidase (Gal) and cephalosporin C acylase (SECA), in comparison with the commonly used Saccharomyces cerevisiae alpha-mating factor pre-pro leader sequence (α-MF) or folding factors (Pdi1p, BiP, and Hac1p). The newly identified signal sequences were superior over α-MF for production of heterologous proteins. The signal peptide Msb2 increased the specific extracellular production of all reporter proteins, ranging from 1.5- to 8.0-fold, and Dan4 enhanced all total protein production up to 172-fold. Co-expression of folding factors exhibited a protein-specific effect on cell growth, transcription and expression of different reporter genes. All of the novel folding factors enhanced total production of SECA, and Sil1p performed best in the extracellular SECA production, showing a 3.3-fold increase. These novel signal peptides and folding factors can be used for promoting secretion of heterologous proteins in P. pastoris.