Improving the Secretory Production of the Heterologous Protein in Pichia pastoris by Focusing on Protein Folding

Abstract

Pichia pastoris has currently been developed as an effective host system for the expression of heterologous genes owing to its potential use for the production of soluble and high-yield proteins. However, the secretory production of the different heterologous proteins in P. pastoris varies widely. Some factors restrict the effective secretory production of heterologous proteins in P. pastoris, among which the folding and processing of proteins is a major one. Besides optimizing the fermentative process, current strategies focus on investigating protein folding process. Thus, this paper is the first time to review the improvement of the secretory production of the heterologous protein in P. pastoris by focusing on its folding process.

Recent advances in the production of recombinant subunit vaccines inPichia pastoris

“…When the recombinant protein fails to fold into its native state or protein expression exceeds the folding capacity of the ER, the unfolded or aggregated proteins may start to accumulate in the ER, thus triggering the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathway. 85 The ER contains several chaperones and foldases such as the hsp70 member Kar2/ Bip, calnexin, calreticulin and protein disulfide isomerase (PDI). 86 The co-overexpression of these ER proteins with the target protein was shown to significantly improve the yields of recombinant proteins.…”

Enhanced expression of heterologous proteins in yeast cells via the modification ofN-glycosylation sites

“…6,10 Both N-and O-linked glycosylation has been reported in yeasts, and the N-linked glycosylation pathway in the ER is conserved between yeasts and most higher eukaryotes. [2][3][4]11 Most N-glycosylation sites are found in putative loop and turn domains of proteins, but a significant number of N-glycosylation sites are present in sites important for the secondary structure of proteins (e.g., b-sheets); approximately, 70-90% of the asparagine (Asn, N) residues in potential N-glycosylation sites are N-glycosylated. N-Glycosylation, which is involved in the process of protein folding in the ER, plays an important role in the production of heterologous secretory proteins requiring N-glycosylation for proper folding.…”

“…1a). [10][11][12] Because N-linked oligosaccharides are large and hydrophilic (e.g., yeasts can produce high mannose-type glycan structures such as Man9GlcNAc2 and have a tendency to hypermannosylate saccharides), their attachment to glycoproteins has the potential to strongly influence the final structure and folding kinetics. 13 Many studies have shown that N-linked glycans can affect or facilitate protein folding in the ER, while their removal from proteins do not affect the biological function, similar to protein chaperones.…”

The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains

“…S. cerevisiae is also an extensively studied host for heterologous protein production and has thus been the subject of much research to engineer and enhance the expression of necessary cellulases to accomplish the ultimate goal of being able to break down lignocellulose for second‐generation production of bioethanol . Despite significant success achieved in cellulase expression in yeast, the secretion titers of cellobiohydrolases (CBHs) have been relatively low, and one of the reasons for this phenomenon is the induction of the unfolded protein response (UPR) .…”

“…Heterologous protein secretion is known to cause the accumulation of unfolded proteins in the endoplasmic reticulum (ER), inducing the UPR with the severity of this response varying with different heterologous genes . The UPR can be described as a highly conserved signaling pathway that is capable of altering gene expression and protein translation to assist with correct folding of proteins during ER stress . The UPR is thus able to signal the nucleus and cytosol information about the protein folding state in the ER lumen, which then buffer the changes caused by the unfolded protein load .…”