Figure 7.

Purification of in vitro-constructed iSAT 70S ribosomes for comparison to purified E. coli 70S ribosomes. (A) Schematic of iSAT 70S ribosome purification. iSAT ribosomes were subjected to ultracentrifugation at 90 000 g for 18 h. The supernatant was removed and the clear ribosome pellet was resuspended in a HEPES-based high salt buffer. Purified iSAT ribosomes were then used in cell-free transcription and translation (TX-TL) reactions. (B) Comparison of sfGFP production by purified iSAT 70S ribosomes and purified E. coli 70S ribosomes. Ribosomes were added to S150 extract-based cell-free TX-TL reactions at a final concentration of 100 nM. Reactions were incubated at 37°C and green fluorescence was measured at 30 min intervals from 0 to 6 h. Values show average sfGFP concentrations above background with error bars representing s.d. for three independent reactions.