Figure 2.
Assessment of PURE and RTS 100 E. coli HY S30 system translation by production of HaloTag fusion proteins using linear DNA templates. HT-Ch-TolA, HT-EntE-Ch-TolA and HT-EntF-Ch-TolA with and without stop codon (82 kD, 141 kD, 224 kD) were synthesized in the PURE system and S30 extract and then incubated with HaloTag TMR Ligand. Equal volume aliquots of samples from PURE system and S30 extract reactions were analyzed on a SDS-PAGE and then scanned by a typhoon scanner with filter set (555nmEx/580nmEm). Lane 1, 3, 5, 8, 10, 12 are HaloTag fusion proteins with stop codon. Lane 2, 4, 6, 9, 11, 13 are HaloTag fusion proteins without stop codon. Lane 7 and 14 are negative controls with no template. (HT: HaloTag, 34 kD; Ch: mCherry, 29 kD. TolA, 19 kD, is a C-terminal 171-amino-acid α-helical spacer excised from E. coli TolA domain II. EntE, 59 kD, and EntF, 142 kD, are multidomain enzymes from E. coli enterobactin biosynthetic pathway.)