Frontier Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, P. R. China.
ACS Synth Biol. 2020 Apr 17;9(4):856-863. doi: 10.1021/acssynbio.9b00508. Epub 2020 Apr 3.
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. Mg 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 Mg: Mg (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; Mg (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 Mg in biological systems.
无细胞蛋白质合成 (CFPS) 是蛋白质工程和合成生物学的一个很有前途的平台。CFPS 系统的储存通常涉及冻干，在此过程中，防止涉及的酶的构象损伤对活性至关重要。本文中，我们报道了镁离子在冻干无细胞系统中对转录和翻译偶联的保护作用。Mg 可防止 CFPS 系统因酶和底物（核苷酸和氨基酸）的直接共冻干而失活，并且还可激活 CFPS 系统。我们提出了 Mg 的双金属离子调控：Mg (I) 作为酶的别构作用，可防止酶在冻干过程中直接与底物结合时的构象损伤，从而锁定无活性的酶-底物复合物；Mg (II) 随后与酶结合以激活 CFPS 系统。我们的工作对于通过在蛋白质工程中使用无细胞系统来最大化蛋白质产量以及理解 Mg 在生物系统中的功能具有重要意义。
