Figure 1.
Expression of insulin chains with ThiS or Ubiquitin fusion?
Insulin A chain (A) or B chain (B) fused with ubiquitin (Ub) or ThiS, were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cells were electrophoresed on 15% SDS-PAGE, shown in left panels. Marker proteins are shown in lane M with sizes at left. Expressed proteins were verified by Western blot probed with anti His-tag antibody, shown in right panels. Arrows highlight expressed proteins at expected positions.
Figure 2.
Expression of Insulin chains fused with half-molecules of ThiS or Ubiquitin.
Insulin A chain (A) or B chain (B) fused with the N-terminal half (ThN-) or C terminal half (ThC-) of ThiS or the N-terminal half of ubiquitin (UbN-), were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cell were resolved on 15% SDS-PAGE, shown in left panels. M indicates Marker proteins. Western blot probed with anti His-tag antibody were shown in right panels. Arrowheads highlight observed positions of expressed proteins.
Figure 3.
Expression of mRI with different tag fusion.
mRI with ubiquitin (Ub) or SUMO fusion were expressed in (A) E. coli TG1 or (B) E. coli BL21 (DE3) pLysS. (C) ThiS fusion of mRI was expressed in E. coli TG1, and protease-deficient strains BL21 (DE3) pLysS, KY2966 and JW3903. (D) mRI with the N-terminal half (ThN-mRI) or C-terminal half (ThC-mRI) of ThiS fusion were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cells were resolved on 10% SDS-PAGE, shown in each upper panel. Western blot probed by anti His-tag antibody was shown in each lower panel. Expressed products migrating at the expected molecular weight are indicated by arrows.
Figure 4.
Enhanced expression of EGFP fused with ThiS.
(A) The recombinant EGFP proteins without (EGFP) or with ThiS-tag (ThiS-EGFP) fused at C-terminus, were induced by 1 mM IPTG for 4 h at 37°C. Total cell lysate (T) and the soluble (S) or insoluble (I) fraction were resolved on 12% SDS-PAGE. Expressed proteins are highlighted by arrows. (B) Expression in total cell lysate from cells at different time after IPTG induction at 37°C were analyzed on 12% SDS-PAGE (upper panel) and immunoblot (lower panel). (C) Cell growth (open circle for EGFP, solid circle for ThiS-EGFP) was recorded by measuring absorbance at 600 nm; the fluorescence of expressed products (open triangle for EGFP, solid triangle for ThiS-EGFP) was measured (excitation 488 nm; emission 509 nm), on different time point after induction by IPTG, for 4 hours at 37°C (the upper panel) or for 20 hours at room temperature (25°C, the lower panel). Each point represents mean and SD of 3 independent experiments. *P<0.05; **P<0.01.
Figure 5.
Folded EGFP proteins with or without ThiS fusion retained their native states during SDS-PAGE.
Purified EGFP and ThiS-EGFP from supernatants (expressed at room temperature), as well as the proteins solubilized from inclusion bodies (I), were resolved on 12% SDS-PAGE, with (+) or without (−) boiling the samples. The gel was photographed under UV illumination (right panel), then stained by Coomassie blue (left panel). Purified native proteins migrated at faster rate and retained fluorescence after SDS-PAGE, if not heat denatured.
Figure 6.
Soluble expression and in vitro refolding of EGFP proteins with or without ThiS fusion.
(A) Three independent clones of E. coli TG1 bearing plasmids expressing EGFP or EGFP fused with ThiS, were induced with IPTG at room temperature for 20 h. Unboiled total cell lysates were resolved on 12% SDS-PAGE. Soluble native form and insoluble denatured form were separated, as indicated by arrows. (B) The native form was verified by UV illumination indicating retained fluorescence of corresponding bands. (C) The Western blot with His-tag antibody further confirmed the identities of overexpressed products. (D) The ratio of native form to unfolded form of ThiS-EGFP was compared to that of EGFP. ** P<0.01. (E) The refolding kinetics of both proteins was compared in vitro. Left panel represents a typical result of the short term refolding curves, with fluorescence (normalized to the respective final fluorescence recovered) plotted against time. In the right panel, kinetics of an initial fast refolding phase, the following slow refolding phase, and the percentage of refolding at final stage (15 h) were compared between two proteins from 3 independent experiments.