Light and heat-shock mediated TDA1 overexpression as a tool for controlled high-yield recombinant protein production in Chlamydomonas reinhardtii chloroplasts
Light and heat-shock mediated TDA1 overexpression as a tool for controlled high-yield recombinant protein production in Chlamydomonas reinhardtii chloroplasts
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DOI：
10.1016/j.algal.2020.101921
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发表时间：
2020-06-01
影响因子：
5.1
通讯作者：
Oey, Melanie
中科院分区：
文献类型：
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作者：
Carrera-Pacheco, Saskya E.;Hankamer, Ben;Oey, Melanie
Microembryonic Chiamyelusreinhardtii为重组蛋白生产提供了一个快速，可扩展和低成本的平台。它的叶绿体提供了一个特别强大的表达系统，用于高产量生产复杂的蛋白质，需要具有挑战性的转录后修饰。受控的转基因表达提供了支持许多应用的重要优势。然而，目前，只有三个诱导系统存在藻类叶绿体表达调控。在这里，我们提出了核编码的翻译增强子TDA 1，调节转基因表达的叶绿体，作为一个组件的诱导系统，是专为高产生产。具体来说，TDA 1的C末端（cTDA 1）通过与其5 '-UTR相互作用来促进atpA转录本的翻译，这已被证明支持高水平的重组蛋白表达。将在诱导型启动子HSP 70 A-RBCS 2控制下的cTDA 1导入在atpA 5 ′-UTR控制下表达GFP的叶绿体突变体中。这些cTDA 1/GFP突变体在优化的光照条件下生长，并进行特定的热休克处理，之后测量cTDA 1和GFP表达水平。在优化的生产条件下生长的诱导样品中检测到类似于1.9倍的增加（在200 μ E.m（-2）.s（-1）的6 h.d（-1）光照下培养4天，然后光照2 h（200 μ E.m（-2）.s（-1））;在40 ℃下热休克30分钟，随后在室温下温育5小时（200 μ E.m（-2）.s（-1）光照）。蛋白质印迹分析证实，诱导后，cTDA 1和GFP的积累相关。这证明了核cTDA 1的表达调节atpA 5 '-UTR介导的叶绿体转基因表达的概念验证，从而为下一代核诱导型叶绿体调节系统提供了基础。
The microalga Chiamydomonas reinhardtii offers a rapid, scalable and low-cost platform for recombinant protein production. Its chloroplast provides a particularly robust expression system for high yield production of complex proteins requiring challenging post-transcriptional modifications. Controlled transgene expression provides an important advantage supporting many applications. Currently, however, only three inducible systems exist for algal chloroplast expression regulation. Here, we present the nuclear encoded translation enhancer TDA1 that regulates transgene expression in the chloroplast, as a component of an inducible system that is designed for high-yield production. Specifically, the C-terminus of TDA1 (cTDA1) promotes translation of the atpA transcript by interacting with its 5'-UTR, which has been shown to support high levels of recombinant protein expression. cTDA1 under the control of the inducible promoter HSP70A-RBCS2 was introduced into a chloroplast mutant expressing GFP under the control of the atpA 5'-UTR. These cTDA1/GFP mutants were grown under optimised illumination regimes and subjected to specific heat-shock treatments, after which cTDA1 and GFP expression levels were measured. A similar to 1.9-fold increase was detected in induced samples grown under optimised production conditions (4 days cultivation period under 6 h.d(-1) illumination at 200 mu E.m(-2).s(-1) followed by 2 h light exposure (200 mu E.m(-2).s(-1)) after the end of the 4 day period; 30 min heat-shock at 40 degrees C with subsequent 5 h incubation at RT (200 mu E.m(-2).s(-1) illumination)). Western Blot analysis confirmed that after induction, the cTDA1 and GFP accumulation correlated. This demonstrates proof-of-concept that expression of the nuclear cTDA1 regulates atpA 5'-UTR mediated chloroplast transgene expression and thus provides the basis for a next generation nuclear inducible chloroplast regulation systems.