Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature

Abstract

Background Escherichia coli heat labile toxin B subunit (LTB) is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. It is often expressed as a fusion partner with target antigens to enhance their immunogenicity as well as gut absorbability. However, high expression levels of a fusion protein are critical to the outcome of immunization experiments and the success of subsequent vaccine development efforts. In order to improve the expression and functional assembly of LTB-fusion proteins using Saccharomyces cerevisiae, we compared their expression under culture conditions at a sub-physiological temperature 20 °C with their expression under a standard 30 °C. Results The assembled expression of LTB-EDIII2 (LTB fused to the envelope domain III (EDIII) of Dengue virus serotype 2), which was expressed at the level of 20 µg/L in our previous study, was higher when the expression temperature was 20 °C as opposed to 30 °C. We also tested whether the expression and functional assembly of a difficult-to-express LTB fusion protein could be increased. The assembled expression of the difficult-to-express LTB-VP1 fusion protein (LTB fused to VP1 antigen of Foot-and-Mouth Disease Virus) dramatically increased, although the total amount of expressed protein was still lower than that of LTB-EDIII2. Slight but significant increase in the expression of well-known reporter protein eGFP, which has previously been shown to be increased by cultivation at 20 °C, was also observed in our expression system. As no significant changes in corresponding transcripts levels and cell growth were observed between 20 °C and 30 °C, we infer that translation and post-translational assembly are responsible for these enhancements. Conclusions The effects of lowering the expression temperature from 30 °C to 20 °C on protein expression and folding levels in S. cerevisiae, using several proteins as models, are reported. When heterologous proteins are expressed at 20 °C, a greater amount of (specially, more assembled) functional proteins accumulated than at 30 °C. Although further studies are required to understand the molecular mechanisms, our results suggest that lowering the expression temperature is a convenient strategy for improving the expression of relatively complexly structured and difficult-to-express proteins in S. cerevisiae.

Combinatorial optimization of pathway, process and media for the production of p‐coumaric acid by Saccharomyces cerevisiae

“…Temperature (T), agitation (rpm) and initial cell density (OD) are usual variables tuned during bioprocess optimization and were selected as factors to improve pCA titers (Couto et al., 2017 ; Duman‐Özdamar et al., 2022 ; So et al., 2023 ). Temperature was varied between 30°C, the optimal growth temperature of S. cerevisiae , and 20°C, as lower temperatures might improve heterologous pathway expression (Table 2 ) (So et al., 2023 ). Agitation and initial OD were varied between 180 and 250 rpm and 0.3–0.6, respectively (Table 2 ).…”

Metabolic engineering of Saccharomyces cerevisiae for the biosynthesis of a fungal pigment from the phytopathogenic fungus Cladosporium phlei

“…The RNA concentration was determined using a Multiskan GO (Thermo Fisher Scientific Inc.). To evaluate the expression levels of the target genes, northern blot analysis and quantitative real-time RT-PCR were performed as previously described [ 40 ].…”

Combinatorial optimization of pathway, process and media for the production of p-coumaric acid bySaccharomyces cerevisiae

“…Temperature (T), agitation (rpm) and initial cell density (OD) are usual variables tuned during bio-process optimization and were selected as factors to improve pCA titers [21][22][23][24][25][26]. Temperature was varied between 30°C, the optimal growth temperature of S. cerevisiae, and 20°C, as lower temperatures might improve heterologous pathway expression (Table 2) [21]. Agitation and initial OD were varied between 180-250 rpm and 0.3-0.6, respectively (Table 2).…”