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

Figure 4

In vitro expression of OGT. (a) Schematic representation of the pBS-rRNA_p-ogt plasmid. It was designed by introducing a DNA fragment of 522 bp containing the ogt gene into the Xho I-Pst I sites replacing ORF 104. The coding region starts with an AUG codon (bold letters) preceding a DNA region coding for six histidines (underlined letters) placed to the amino terminal region of the OGT protein (bold and italic letters). The DNA insert contains an SD motif (italic letters) retained from the ORF 104 and located 7 nucleotides upstream from the coding region. (b) Increased amounts of the pBS-rRNA_p-ogt plasmid were incubated with S. solfataricus whole cell extract for 60 min at 70°C in a final volume of 25 μl, and the products of expression were resolved by 16% denaturing polyacrylamide gel electrophoresis. (c) Time course of OGT expression: 4 μg of pBS-rRNA_p-ogt plasmid were incubated with S. solfataricus whole cell extract at 70°C and equal aliquots of the reaction were withdrawn from the mixture at the indicated times. (d) A graph is plotted with the values of the band intensity corresponding to the OGT protein shown in (c) and quantified using ImageJ software (NIH). The values represent the average of three independent experiments. All error bars indicate SD.

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