Wang Yanan, Wang Ting, Chen Xinjie, Lu Yuan
Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, Beijing, 100084, China.
Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.
Bioresour Bioprocess. 2023 Jun 20;10(1):35. doi: 10.1186/s40643-023-00653-4.
Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K and Mg) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.
无细胞蛋白质合成（CFPS）系统是用于快速便捷蛋白质研究的理想平台，已被用于大分子组装、非天然氨基酸嵌入、糖蛋白生产等。为了构建一个具有低成本和短时间优势的高效真核CFPS平台，本研究构建了基于毕赤酵母的CFPS系统。内部核糖体进入位点（IRES）可独立启动翻译，从而促进蛋白质合成。科扎克序列可促进翻译起始。因此，对IRES及其与科扎克序列的组合进行了筛选，其中蟋蟀麻痹病毒（CRPV）在14种不同的IRES中表现为最佳翻译起始元件。此外，还对系统组件和反应环境进行了探索。添加核糖核酸酶抑制剂后蛋白质产量几乎翻倍。对细胞提取物量、能量再生系统（磷酸肌酸和磷酸肌酸激酶）和金属离子（钾和镁）进行了优化，以实现最佳蛋白质合成产量。这种毕赤酵母CFPS系统可以扩展真核CFPS平台，为快速原型设计和功能性蛋白质合成提供一项赋能技术。
