Many researchers prefer using Pichia pastoris, a strain of yeast, to model the production and secretion of recombinant proteins. Use of the yeast secretory pathway bestows several benefits, including the avoidance of toxic intracellular materials, easier purification of the product, and simplified opportunity for the occurrence of post-translational events. The P. pastoris system, however, can become overburdened by the folding and post-translational modifications that occur in the endoplasmic reticulum (ER), resulting in rate and yield limitations. Evidence of this overburden includes the non-proportional ratio of secreted proteins to gene copies, mRNAs or intracellular proteins. Other reported signs of overuse include ER dilation, intracellular aggregation, and protein congregation with chaperones and transcription factor Hac1p. Studying this stress response may allow researchers to engineer host cells that resist stress and result in greater protein yields.
In this study, Lin et al. (2013) utilized xylanase A from Bacillus halodurans C-125 (xynA) to evaluate whether high levels of protein secretion induce an unfolded protein response (UPR) in the ER. To do this, the researchers overexpressed both xynA and HAC1 and found that employing this genetic modification increased the secretion rate of xylanase A. The researchers examined the link between xynA gene copy number and protein expression in P. pastoris through a comparative analysis among four transgenic strains of the yeast: a control strain with plasmid pPICZaA that lacked the xynA gene (G0); a low-expression strain with one copy of xynA (G1); a high-expression strain with four copies of the plasmid (G4); and a high xynA expression strain that also expressed HAC1 (G4-H). Lin et al. demonstrated a 3-fold increase in xylanase A secretion in strain G4 as compared to strain G1. Between strains G4 and G4-H, a 1.4-fold increase was observed in the G4-H strain. In terms of transcription, strain G4 evidenced a 2.59-fold increase and strain G4-H a 2.8-fold increase when compared to strain G1.
The researchers then turned to high-throughput chromatography with mass spectrometry, utilizing an 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) method for labeling to ascertain if the increased protein expression occurred as a result of upregulation of UPR-driven transcription factors. They relied upon Proteome Discoverer (Thermo Scientific) for data processing. In this way, Lin et al. obtained 15,225 unique spectra out of 227,112 total spectra. These unique spectra represented 1,210 unique proteins and included 352 proteins that demonstrated statistically significant variance among the four strains of P. pastoris. The researchers also noted the upregulation of 10 proteins intrinsic to the protein folding process (Sar1p, Sec61p, Cne1p, Pdi1p, Sec53p, Lhs1p, Sss1p, Swp1p, Wbp1p, chr1-1_0459) in strains G4 and G4-H. There was also significant downregulation of ribosomal proteins in the G4-H strain.
Overall, Lin et al. found that excessive expression and secretion of xylanase A improved protein production but that the subsequent burden of this overproduction also induced UPR in the ER. The researchers also found that co-expression of the transcription factor Hac1p addressed this issue by downregulating protein synthesis while upregulating the efficiency of protein folding in the ER.
Lin, X., et al. (2013) “Quantitative iTRAQ LC–MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris,” Journal of Proteomics, http://dx.doi.org/10.1016/j.jprot.2013.06.031.
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.