This study described the mining of a library of engineered yeast strains modified by overexpression of endogenous yeast proteins. It was discovered that although yeast surface display allowed rapid quantitative sorting of the engineered strains, the Aga2p tether masked the effects that the overexpressed yeast proteins had on the scFv or scTCR fusion partner. However, one of the proteins (7/15 scTCR) was particularly sensitive to induction temperature. Thus, under the influence of an elevated temperature that decreased the efficiency of intracellular processing, several yeast strains that promoted increased display and secretion were isolated. The increases were mediated by overexpression of translational components (coded for by RPP0), ER-resident folding assistants (coded for by ERO1), and cell wall proteins (coded for by SED1, CCW12, and CWP2), few of which would likely have been predicted a priori. The increases in heterologous protein secretion were not limited to the screened scTCR, but were also generalizable to additional scTCR and scFv proteins.
Although secreted protein and Aga2p fusion protein destined for display on the cell surface both traverse the same secretory compartments, our observations indicated that the association with the cellular folding machinery, such as BiP and PDI, differed substantially. In particular, increasing the expression levels of the ER-resident BiP and/or PDI had already proven successful in increasing secretion of scFv and scTCR from S. cerevisiae
(this work and references 9
, and 35
). However, BiP and PDI overexpression had no effect on surface display levels. In addition, although an scFv and two scTCR that differ 20-fold in secretion efficiency were put through the initial selection strategy without selection pressure, no overexpressed yeast proteins that could increase display levels were identified. Thus, it appeared that fusion to the Aga2p display scaffold enabled the scFv to bypass the intracellular bottleneck normally encountered by unfused scFv. In addition, even when scFv display was induced at elevated temperatures that normally diminish secretion titers (9
), the display levels were not affected, again indicating that Aga2p could dominate the display efficiency of its scFv fusion partner. The consequence of these findings is that screens for engineered yeast cannot be performed under conditions where Aga2p dominates display efficiency.
Thus, to overcome the dominant effects of Aga2p and allow the yeast strain engineering approach to identify yeast proteins that can enhance display and secretion, we employed an scTCR protein whose display levels, unlike those of scFv, were particularly responsive to a selection pressure of elevated induction temperature. In this way, five yeast genes that restore or increase display levels of active protein were identified. Although the five yeast genes increased display of the low-stability 7/15 scTCR at the elevated temperatures, only ERO1 overexpression increased display levels at 20°C (1.4-fold increase), again suggesting that the selection pressure was required to select CCW12, RPP0, SED1, and CWP2 from the yeast library due to Aga2p masking effects. Therefore, although it would be ideal to use this system to screen engineered yeast libraries for any heterologous protein of interest, the protein of interest must be responsive to a selection pressure such as elevated induction temperature for the display-based screen to be successful. However, display-based screening with a single protein substrate allowed the identification of five yeast proteins, several of which can serve as fairly generalizable secretion assistants as discussed below.
Although the five yeast genes were selected at higher temperatures, all except CWP2 promoted increased secretion of at least one protein at 20°C, and 20°C proved optimal for the maximum secretion levels. The two scFvs tested behaved similarly to the 7/15 scTCR in that CCW12 and ERO1 could enhance secretion, albeit to different extents. However, unlike 7/15, the scFvs exhibited increased secretion levels in response to RPP0 overexpression. In contrast to the products of these three genes, the ultrastable LWHI scTCR did not respond to any of the overexpressed yeast genes other than showing modest increases with ERO1 overexpression. Taken together, the temperature-dependent display enhancers (CCW12, CWP2, SED1, and RPP0) seem to facilitate secretion of the lower-expression/stability proteins, while secretion of the LWHI protein was unaffected. On the other hand, the lone 20°C display enhancer, ERO1, yielded statistically significant increases in secretion for all proteins tested, indicating that the most general solutions would be those selected under conditions of induction at 20°C. Unfortunately, as discussed above, the presence of Aga2p prevents such direct selections from being successful.
Two of the isolated display enhancers, Ero1p and Rpp0p, are known to function directly in the protein synthesis and folding process and were therefore expected to enhance protein secretion. The Ero1p protein is essential for yeast viability and functions in delivering oxidizing equivalents to folding disulfide-containing proteins through PDI (5
is induced by the unfolded protein response and loss of Ero1p results in accumulation of reduced protein in the ER (5
). Therefore, since each of the heterologous proteins investigated contains two disulfide bonds, overexpression of Ero1p likely assists in the formation of these disulfide bonds and promotes exit from the ER. For example, overexpression of Kluveromyces lactis ERO1
has led to increased secretion of disulfide-bonded human serum albumin, but not disulfide-free interleukin-1β (18
). The P0 protein (Rpp0p) is one of a set of proteins that assemble at the stalk of the large ribosomal subunit in yeast (20
), and excess Rpp0p is not normally observed (31
). Thus, it may be possible that under conditions of heterologous protein overexpression, the Rpp0p protein may be a limiting component in the ribosomal assembly, and this deficiency in protein translation capacity may be alleviated by overexpression of the Rpp0p protein. Alternatively, Rpp0p may be functioning indirectly as overexpressed Rpp0p has been implicated in alleviating prion formation in yeast by increasing the activity of promoters containing heat shock elements that drive expression of many chaperones and foldases (15
We initially hypothesized that several of the genes recovered in the library screen, namely CCW12, CWP2, and SED1, might not increase secretion of the unfused 7/15 scTCR, as these genes have cellular functions related to the yeast cell wall. Since the flow cytometry selection process required surface display involving the Aga1p and Aga2p cell wall proteins, the recovered clones could have been the result of “you get what you select for,” and yeast proteins that facilitate Aga1p and/or Aga2p assembly and processing, rather than scTCR processing, could have been selected. However, none of the cell wall proteins, when overexpressed, affected the display of Aga2p lacking the scTCR fusion partner. Thus, it appeared that the cell wall proteins were regulating surface display in an scTCR-dependent manner and may have had a general influence on the secretory processing of scTCR. Indeed, overexpression of the cell wall genes increased both the surface display of 7/15 (CCW12, CWP2, and SED1) and the secretion of 7/15 (CCW12). scFv secretion was also elevated by CCW12 overexpression, and to a lesser extent by CWP2 and SED1. In contrast, the LWHI scTCR was unaffected by cell wall protein expression, indicating protein-specific effects and not a general change in cell physiology.
Each of the cell wall proteins is covalently linked to the cell wall glycan layer after processing as a glycosylphosphatidylinositol-anchored precursor (21
). The proteins have generally been implicated in providing cell wall stability and resistance to stresses. For example, CCW12
deletion or overexpression increases the sensitivity to known cell wall perturbants calcofluor white and Congo red (21
), deletion of SED1
made stationary-phase cells more sensitive to Zymolyase treatment (32
), and deletion of CWP2
, like CCW12
, increased sensitivity to calcofluor white and Congo red while also increasing the sensitivity of exponentially growing cells to Zymolyase treatment (42
). Thus, the stresses imposed by heterologous protein display and secretion may be diminished by overexpression of cell wall proteins. Although further study will be required to elucidate the mechanism whereby the cell wall proteins assist secretion and display, the results of this study clearly point to the cell wall as a novel target for secretion improvement.