Institute for Molecular Biology and Genetics, Department of Molecular Biology, Jeonbuk National University, Jeonju, Jeollabuk-Do, 54896, Republic of Korea.
Department of Bioactive Material Sciences, Jeonbuk National University, Jeonju, Jeollabuk-Do, 54896, Republic of Korea.
Microb Cell Fact. 2023 Mar 23;22(1):55. doi: 10.1186/s12934-023-02065-7.
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.
The assembled expression of LTB-EDIII (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-EDIII. 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.
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.
大肠杆菌不耐热肠毒素 B 亚单位（LTB）是最受欢迎的口服疫苗佐剂和肠道吸附增强剂之一。它通常被表达为与靶抗原的融合伙伴，以增强其免疫原性和肠道吸收性。然而，融合蛋白的高表达水平对免疫实验的结果和后续疫苗开发工作的成功至关重要。为了提高酿酒酵母中 LTB 融合蛋白的表达和功能组装，我们比较了在亚生理温度 20°C 下与在标准 30°C 下培养条件下的表达。
在我们之前的研究中，在 20°C 下表达水平为 20μg/L 的 LTB-EDIII（与登革热病毒血清型 2 的包膜域 III（EDIII）融合的 LTB）的组装表达更高。我们还测试了是否可以增加难以表达的 LTB 融合蛋白的表达和功能组装。难以表达的 LTB-VP1 融合蛋白（与口蹄疫病毒 VP1 抗原融合的 LTB）的组装表达显著增加，尽管表达的总蛋白量仍低于 LTB-EDIII。以前已经表明在 20°C 培养时会增加的已知报告蛋白 eGFP 的表达也略有但明显增加，在我们的表达系统中也观察到了这种情况。由于在 20°C 和 30°C 之间没有观察到相应转录物水平和细胞生长的显著变化，我们推断翻译和翻译后组装负责这些增强。
使用几种蛋白质作为模型，报告了从 30°C 降低到 20°C 对酿酒酵母中蛋白质表达和折叠水平的影响。当异源蛋白在 20°C 下表达时，与在 30°C 下相比，积累了更多的（特别是更多组装的）功能性蛋白质。尽管需要进一步研究来了解分子机制，但我们的结果表明，降低表达温度是提高酿酒酵母中相对复杂结构和难以表达的蛋白质表达的一种便捷策略。
