Figure 5

Luc, eGFP and eGFP-Del6-229 production as a function of temperature. (A) End-point measurement of Luc production as a function of PEG 8000 concentration and as a function of the reaction incubation temperature (3-PGA buffer, conditions: 0 mM magnesium glutamate, 80 mM potassium glutamate, 1.5 mM of each amino acid). (B) End-point measurement of eGFP and eGFP-Del6-229 production as a function of the reaction incubation temperature (3-PGA buffer, conditions: 4 mM magnesium glutamate, 60 mM potassium glutamate, 1.5 mM of each amino acid, 2.5% PEG 8000). (C) Temperature stability of eGFP and eGFP-Del6-229 protein expressed with 2 nM plasmids (pBEST-UTR1-eGFP-T500 and pBEST-UTR1-eGFP-Del6-229-T500). 1) 8 hours expression of eGFP at 32°C, 2) 8 hours expression of eGFP-Del6-229 at 32°C, 3) 8 hours expression of eGFP at 29°C, 4) 8 hours expression of eGFP-Del6-229 at 29°C, 5) 8 hours expression of eGFP at 32°C followed by 1 hour of incubation at 37°C, 6) 8 hours expression of eGFP-Del6-229 at 32°C followed by 1 hour of incubation at 37°C, 7) 8 hours expression of eGFP at 29°C followed by 1 hour of incubation at 37°C, 8) 8 hours expression of eGFP-Del6-229 at 29°C followed by 1 hour of incubation at 37°C. 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.