Figure 4
From: Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70
Analysis of protein production by polyacrylamide gel electrophoresis. For both gel, lane 1 is a negative control (no plasmid added in the reaction), lane 2 and 6 is a protein ladder (5 main bands from top to bottom 75, 50, 37, 25 and 20 kDa). Synthesized proteins are indicated by arrows. (A) Luc synthesized with 10 nM pBEST-Luc plasmid (lane 3), 1.5 nM pBEST-UTR1-Luc plasmid (lane 4), and 15 nM pBEST-UTR1-Luc plasmid (lane 4) (3-PGA buffer, conditions: 1 mM magnesium glutamate, 60 mM potassium glutamate, 1.5 mM of each amino acid, 0.5% PEG 8000). Pure Luc added to the cell-free reaction with no plasmid, 2 μM (lane 7), 6 μM (lane 8), 10 μM (lane 9) and 14 μM (lane 10). (B) eGFP synthesized with 15 nM pBEST-eGFP plasmid (lane 3), 1 nM pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 plasmid (lane 4) and 10 nM pBEST-OR2-OR1-Pr-UTR1-eGFP-Del6-229-T500 plasmid (lane 5), (3-PGA buffer, conditions: 3 mM magnesium glutamate, 30 mM potassium glutamate, 1.5 mM of each amino acid, 2% PEG 8000). Pure eGFP added to the cell-free reaction with no plasmid, 10 μM (lane 7), 15 μM (lane 8), 20 μM (lane 9) and 25 μM (lane 10). Concentrations of magnesium glutamate and potassium glutamate reported here are the concentrations added to the cell-free reaction. The crude extract, dialyzed against the S30 buffer B, brings an additional 4.5 mM magnesium glutamate and 20 mM potassium glutamate to the cell-free reaction.