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. 2020 Oct 5;295(50):17265–17280. doi: 10.1074/jbc.RA120.015557

Figure 4.

Figure 4.

Effect of optimized tRNA and condensed culture strategy on amber suppression efficiency. Lanes 1–4 contain samples from E. coli harboring pcIts-(T7 G5x− E514TAG, trxA) and pSUP-(CouRS-6TRN), analyzed by SDS-PAGE. Lanes 5–10 contain samples from E. coli harboring pcIts-(T7 G5x− E514TAG, trxA) and pSUP-(CouRS-tRNAopt), analyzed by SDS-PAGE. Samples with a + in the row labeled 10 × Cond. were condensed 10× by gentle centrifugation of the cells and resuspension into media with 1 mm 7-HCou before inducing protein expression. The row labeled 7-HCou indicates whether 7-HCou was added to the expression media (+), or not added to the expression media (−) to test for background incorporation of naturally occurring amino acids. The row labeled Ind. indicates whether the cells were collected before inducing protein expression (Pre) or 3 h after inducing protein expression (Post). The arrow to the right of the gel indicates the position of the band for T7 GP5 at ∼80 kDa.