Continuous translation of circularized mRNA improves recombinant protein titer
Continuous translation of circularized mRNA improves recombinant protein titer
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DOI：
10.1016/j.ymben.2019.01.002
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发表时间：
2019-03-01
影响因子：
8.4
通讯作者：
Clynes, Martin
中科院分区：
文献类型：
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作者：
Costello, Alan;Lao, Nga T.;Clynes, Martin
最近在证明环状RNA开放阅读框或环状mRNA的翻译方面的成功，可能为改善来自细胞和无细胞表达平台的重组蛋白生产提供新的途径。起始和终止是翻译的两个限速步骤。环状RNA作为一类RNA通过末端共价连接以给出闭环结构来定义。通过编码环状RNA分子上缺少终止密码子的基因，产生允许连续翻译环状mRNA（CTC mRNA）的无限开放阅读框。CTC mRNA在细菌无细胞表达系统中增强多聚体多蛋白的产生方面已显示出前景。当需要同质的功能性后修饰蛋白质时，问题出现了。为了从CTC mRNA产生后修饰的分泌蛋白，我们研究了通过掺入2A“自切割”肽基序对新生多肽链的共翻译切割。使用模型重组人糖蛋白促红细胞生成素（EPO），我们证明了第一次在活的哺乳动物细胞中从无限环状mRNA产生分泌蛋白的能力。通过使用环状mRNA，细胞特异性和体积生产率都得到了改善。这项研究开创了在哺乳动物细胞中从CTC mRNA生产重组蛋白的潜力。
Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A "self-cleavage" peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.