Cell-free protein synthesis (CFPS) is a promising platform for protein engineering and synthetic biology. The storage of a CFPS system usually involves lyophilization, during which preventing the conformational damage of involved enzymes is critical to the activity. Herein, we report the protection role of magnesium ions on coupled transcription and translation in a lyophilized cell-free system. Mg2+ prevents the inactivation of the CFPS system from direct colyophilization of enzymes and substrates (nucleotides, and amino acids), and furthermore activates the CFPS system. We propose two-metal-ion regulation of Mg2+: Mg2+ (I) acts as an allosteric role for enzymes to prevent the conformational damage of enzymes from direct binding with substrates during lyophilization which locks up inactive enzyme-substrate complex; Mg2+ (II) consequently binds to enzymes to activate the CFPS system. Our work provides important implications for maximizing protein yields by using a cell-free system in protein engineering and understanding the functions of Mg2+ in biological systems.

中文翻译：

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镁离子对冻干无细胞系统中转录和翻译偶联的保护作用。

无细胞蛋白质合成（CFPS）是蛋白质工程和合成生物学的有前途的平台。CFPS系统的存储通常涉及冻干，在冻干过程中，防止所涉及的酶的构象破坏对活性至关重要。在此，我们报道了镁离子在冻干的无细胞系统中对偶联的转录和翻译的保护作用。Mg2 +可以防止酶和底物（核苷酸和氨基酸）直接共冻干而使CFPS系统失活，并进一步激活CFPS系统。我们提出了Mg2 +的两种金属离子调节：Mg2 +（I）充当酶的变构作用，以防止酶的构象损伤在冻干过程中直接与底物结合，从而锁定了无活性的酶-底物复合物。因此，Mg2 +（II）与酶结合以激活CFPS系统。通过在蛋白质工程中使用无细胞系统并了解Mg2 +在生物系统中的功能，我们的工作对最大程度地提高蛋白质产量具有重要意义。