The protection role of magnesium ions on coupled transcription and translation in lyophilized cell-free system.

Cell-free protein synthesis (CFPS) is a promising platform for protein engineering and synthetic biology. The storage of 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 lyophilized cell-free system. Mg2+ prevents the inactivation of the CFPS system from direct co-lyophilization of enzymes and substrates (nucleotide, amino acids and cofactors), 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 implication for maximizing protein yields by using cell-free system in protein engineering and understanding the functions of Mg2+ in biological systems.