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Using a viral 2A peptide-based strategy to reconstruct the bovine P450scc steroidogenic system in S. cerevisiae
Journal of Biotechnology ( IF 4.1 ) Pub Date : 2020-11-03 , DOI: 10.1016/j.jbiotec.2020.10.028
Vera S Efimova 1 , Ludmila V Isaeva 2 , Philipp S Orekhov 3 , Marine E Bozdaganyan 4 , Mikhail A Rubtsov 5 , Ludmila A Novikova 2
Affiliation  

Cytochrome P450scc system performs the first rate-limiting stage of steroidogenesis in mammals. The bovine P450scc system was reconstructed in Saccharomyces cerevisiae, using a foot-and-mouth disease virus 2A peptide (F2A)‐based construct, to co-express cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). During the translation of the self‐processing fusion protein P450scc-F2A-Adx-F2A-AdR, the first and the second linkers are cleaved with different efficiencies (96 % and 11 %, respectively), resulting in the unbalanced expression of individual proteins. The low cleavage efficiency and the relative Adx and AdR protein levels were increased through replacing the second F2A peptide with different sequences and changing the order of Adx and AdR. The P450scc, AdR, and Adx sequences located upstream of the F2A affected F2A processing, to various degrees. Moreover, using molecular dynamics (MD) simulations, we showed that the 2A peptide fused to the C-terminus of Adx formed the steric hindrance during enzymatic complex formation, resulting in the reduction of catalytic activity. Thus, the functional activity of the reconstructed P450scc system was determined not only by the efficiency of 2A peptides but also by the overall sequence of the expressed 2A-polyprotein. Our results can be applied to the development of 2A-based co-translation strategies, to produce other multicomponent protein systems.



中文翻译:

使用基于病毒 2A 肽的策略重建酿酒酵母中的牛 P450scc 类固醇生成系统

细胞色素 P450scc 系统执行哺乳动物类固醇生成的第一个限速阶段。牛 P450scc 系统在酿酒酵母中重建使用基于口蹄疫病毒 2A 肽 (F2A) 的构建体,共表达细胞色素 P450scc、肾上腺素氧还蛋白 (Adx) 和肾上腺素氧还蛋白还原酶 (AdR)。在自加工融合蛋白 P450scc-F2A-Adx-F2A-AdR 的翻译过程中,第一个和第二个接头以不同的效率(分别为 96% 和 11%)被切割,导致单个蛋白质的不平衡表达。通过用不同序列替换第二个 F2A 肽并改变 Adx 和 AdR 的顺序,提高了低切割效率和相对 Adx 和 AdR 蛋白水平。位于 F2A 上游的 P450scc、AdR 和 Adx 序列在不同程度上影响了 F2A 处理。此外,使用分子动力学 (MD) 模拟,我们发现与 Adx 的 C 端融合的 2A 肽在酶复合物形成过程中形成空间位阻,导致催化活性降低。因此,重建的 P450scc 系统的功能活性不仅取决于 2A 肽的效率,还取决于表达的 2A 多聚蛋白的整体序列。我们的结果可用于开发基于 2A 的共翻译策略,以产生其他多组分蛋白质系统。

更新日期:2020-11-03
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