Figure 1.

Improvement of colicin M production in cell-free protein synthesis (CFPS). (A) Total and soluble protein yield for cell-free produced colicin M with molecular chaperone-enriched extracts quantified by ¹⁴C-Leu scintillation counting at 30 °C and room temperature (RT). Cell extract without chaperone production was prepared from BL21 Star (DE3) strain (Star). Chaperone-enriched extracts were prepared from BL21 Star (DE3) cultures overexpressing GroES/EL (Gro), DnaK/DnaJ/GrpE (KJE), and both GroES/EL and DnaK/DnaJ/GrpE (Gro-KJE) in BL21 Star (DE3). Error bars indicate standard deviations from three independent CFPS reactions. (B) Radioactive ¹⁴C-Leu autoradiogram gel of total (T) and soluble (S) protein yield for colicin M and sfGFP produced during CFPS reactions with different cell extracts. (C) Viability of K361 indicator cells. K361 cells (initial cell density 5 × 10⁷ CFU/mL) were treated with 750 ng/mL total concentration of colicin M produced by CFPS using different chaperone-enriched extracts and then incubated at 37 °C and 220 rpm for 1 h. Error bars indicate standard deviation from two independent cultures with three plating replicates each. ** and *** represent significant difference compared to no addition sample under the same media conditions with p-values of < 0.001 and <0.0001, respectively.