Fig 1.
A schematic overview of the cell-free protein production process.
Table 1.
Preparation of S30-T7 lysate.
Table 2.
In vitro protein synthesis using cell-free system based on S30-T7 lysate.
Table 3.
The composition of the CFPS reaction mixture.
Fig 2.
A historical overview of improvements made to CFPS procedures over time.
Fig 3.
Enzyme productions using S30-T7 CFPS systems sourced from two different E. coli strains (BL21 and MRE600).
(A) The produced Renilla luciferase activity was demonstrated by integrating 10 seconds of luminescence measurements (error bars represent standard deviation from at least three independent samples). (B) & (C) TyrBm production was confirmed by monitoring the conversion of 1mM L-Dopa to dopachrome (error bars represent standard deviation from three independent samples). (C) The observed enzymatic activity of TyrBm, produced by the S30-T7 CFPS in a 96-well plate. The three wells to the right present cell-free reaction in the present of DNA template, while in the three wells to the left no DNA template was incorporated into the reaction. The dark color indicates on the conversion of L-Dopa to dopachrome (followed by polymerization and accumulation of melanin), and thus on the production of TyrBm. (D) Temperature effect on cell-free superfolder GFP production efficiency of the S30-T7 CFPS (error bars represent standard deviation from at least four independent samples). The protein production amount was evaluated according to the fluorescence levels. The fluorescence values obtained at 37°C were set to 100%, and all the other values were normalized according to them. Negative controls (N.C.) were reactions without DNA templates. * Significant difference between lysates from the two E. coli strains, where α<0.05 according to a Student's t-Test with a two-tailed distribution with equal variance.
Fig 4.
Pseudomonas exotoxin (PE) productions using the S30-T7 CFPS system originated from two different E. coli strains (BL21 and MRE600).
Reactions were performed with and without the presence of DNA template. (A) Western blot analysis of cell-free reactions demonstrated the production of PE ~ 66 kDa. Purified PE served as positive control (described in Appendix F in S1 File.). Arrows indicate the position of PE bands. (B) The therapeutic potency of PE was evaluated on 4T1 cell-line. The viability of the cells was determined by MTT assay. Cell viability values obtained without the presence of purified PE or DNA were set as 100%, and the other values were normalized according to them (error bars represent standard deviation from at least three independent samples).