Figure 2
From: High yield expression of catalytically active USP18 (UBP43) using a Trigger Factor fusion system
Expression of TF AAA -USP18 in pSUMO vector backbone under different conditions (A) TF AAA -USP18 was expressed in E. coli BL21(DE3)pLysS at 37°C. Expression was verified by analyzing protein content directly after lysis on SDS-PAGE followed by Coomassie staining. After 3 h of induction TFAAA-USP18 fusion protein made up more than 50% of whole cellular proteins. (B) Soluble and insoluble fractions from (A) were analysed by Western blot with an anti His6-Tag antibody. Almost all fusion protein was present in the insoluble fraction and only a faint band for soluble protein was observed. (C) Expression of TFAAA-USP18 at 15°C in E. coli BL21(DE3)pLysS yielded soluble protein. Western blot analysis using an anti His6-Tag antibody detected TFAAA-USP18 only in the soluble fraction. (D) E. coli Tuner(DE3) and E. coli Tuner(DE3)pLysS were tested for expression of TFAAA-USP18 at 15°C. Soluble and insoluble fractions were analysed by SDS-PAGE and subsequent Coomassie staining. Strong expression was only observed in E. coli Tuner(DE3). The major portion of the fusion protein was observed in the soluble fraction.