Extended Data Fig. 6: Incorporation of XisoK derivatives using the IsoK12 strain.
From: Hijacking a bacterial ABC transporter for genetic code expansion
a. SDS-PAGE analysis of sfGFP-N150TAG expression in 2-YT, comparing K12 with IsoK12 cells at different G-SisoK concentrations. Full-length sfGFP levels at 0.1 mM G-SisoK in IsoK12 are comparable to those obtained with 1 mM G-SisoK in wt-K12. b. Top: SDS-PAGE analysis of amber suppression of proliferating cell nuclear antigen (PCNA-K164TAG), heat shock protein 82 (Hsp82-D452TAG), calmodulin (calmodulin-G40TAG) and interleukin-2 (IL-2-R38TAG) with 1 mM G-SisoK in wt-K12 or IsoK12 cells grown in 2-YT. The expression of full-length proteins is higher in IsoK12 strain compared to wt-K12. Bottom: SDS-PAGE analysis of amber suppression of RanGTPase activating protein (RanGAP-K524TAG) and human growth hormone (hGH-Y35TAG and hGH-K38TAG) expressed with 1 mM BocK or G-SisoK in IsoK12 cells grown in 2-YT. Full-length protein expression levels are higher with G-SisoK in comparison to BocK. c. Left: SDS-PAGE analysis of eGFPNb-R17TAG expression in the presence of 1 mM G-PrgisoK or PraK59 in AI or 2-YT media comparing wt-K12 with IsoK12. Full-length eGFPNb expression is highest in IsoK12 cells grown in 2-YT. Right: Preparative protein yields of wt-eGFPNb, eGFPNb-R17PrgisoK or eGFPNb-R17PraK. wt-MbPylRS/PylT pair was used for PraK and G-PrgisoK incorporation. PrgisoK-bearing eGFPNb yields exceed yields for PraK-bearing eGFPNb, matching wt-eGFPNb yields. For structure of PraK see Extended Data Fig. 3a. d. SDS-PAGE analysis of Histone H3 expression with single (K122TAG), double (K79TAG, K122TAG) and triple (K27TAG, K79TAG, K122TAG) amber suppression in presence of 1 mM G-SisoK comparing K12 with IsoK12 cells grown in 2-YT media. a-d. Consistent results were obtained over three distinct replicate experiments. Arrows indicated full-length POIs, asterisks indicated truncated POIs.
