Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Figure 5

In vitro expression of ogt from linearized plasmid. (a) Supercoiled and linear pBS-rRNA_p-ogt plasmids were incubated with S. solfataricus whole cell extract for 60 min at 70°C with ³⁵S-Met in a final volume of 25 μl, and the products of expression were resolved by 16% denaturing polyacrylamide gel electrophoresis. (b) Survival of supercoiled and linear pBS-rRNA_p-ogt plasmid after incubation in the S30-coupled system. The constructs were incubated for 60 min at 70°C under standard conditions and then analysed on a 1% agarose gel. Lane 1, nonincubated linear pBS-rRNA_p-ogt DNA; lane 2, nonincubated supercoiled pBS-rRNA_p-ogt DNA; lane 3, linear pBS-rRNA_p-ogt DNA incubated in an S30 mixture; and lane 4, supercoiled pBS-rRNA_p-ogt DNA incubated in an S30 mixture.

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