Fig 1.
SDS-PAGE analysis of the expression of fusion protein ACP-GlcK (panel A) and ACP-Amy (panel B) in E. coli strain BL21 (DE3).
After induction by addition of IPTG to 0.4 mM in the medium at 20°C for 12 h, E. coli cells were harvested. After sonication, the cell lysate was centrifuged to remove cell debris, then the supernatants were recovered and the insoluble fraction was assumed to consist of inclusion bodies. W: whole cell lysate; S: supernatant; I: inclusion bodies; +: IPTG was added to culture medium to induce the expression of recombinant proteins; -: no IPTG was added.
Fig 2.
Purification of the passenger proteins (target proteins) by two consecutive affinity chromatography steps.
Recombinant fusion proteins were first purified by Ni-chelating affinity chromatography. After that, the fusion proteins were subjected to processing in vitro by mixing them with the purified His-tagged protease TEV. Lastly, the resulting target proteins were separated by additional Ni-chelating affinity chromatography to remove His-tagged protease TEV and the fusion tag ACP. All samples were subjected to 12% SDS-PAGE analysis, lane 1, purified fusion protein Histag-ACP-GlcK; lane 2, the purified fusion protein Histag-ACP-GlcK cleaved in vitro by pure His-tagged protease TEV; lane 3, purified fusion protein Histag-ACP-G2-GlcK; lane 4, the purified fusion protein Histag-ACP-G2-GlcK cleaved in vitro by pure His-tagged protease TEV; lane 5, purified target protein GlcK; lane 6, purified fusion protein Histag-ACP-Amy; lane 7, the purified fusion protein Histag-ACP-Amy cleaved in vitro by pure His-tagged protease TEV; lane 8, purified fusion protein Histag-ACP-G2-Amy; lane 9, the purified fusion protein Histag-ACP-G2-Amy cleaved in vitro by pure His-tagged protease TEV; lane 10, purified target protein Amy.
Fig 3.
Intracellular processing of fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag by the protease TEV coexpressed in E. coli and subsequently purification of the target protein GlcK and Amy by one-step Ni-chelating chromatography.
The clarified supernatants of E. coli cell lysate and purified proteins were subjected to 12% SDS-PAGE analysis, lane 1 and lane 6, the supernatant of the cell lysate of E. coli expressing fusion proteins; lane 2 and lane 7, the supernatant of the cell lysate of E. coli expressing fusion proteins treated by addition of the purified protease TEV; lane 3 and lane 8, the supernatant of the cell lysate of E. coli coexpressing the fusion proteins with protease TEV, which was expressed by vector pBAD34-MBP-TEV-WHZ; lane 4 and lane 9, the supernatant of the cell lysate of E. coli coexpressing the fusion proteins with protease TEV, which was expressed by vector pSU18-MBP-TEV-WHZ; lane 5 and lane 10, purified target proteins.
Fig 4.
Intracellular processing of the fusion protein ACP-GFP-Histag by the protease TEV coexpressed in E. coli.
E. coli cells were transformed with vector pSU18-MBP-TEV-WHZ and pET-28b-ACP-GFP-Histag respectively, or co-transformed with vector pSU18-MBP-TEV-WHZ and pET-28b-ACP-GFP-Histag. After induced by addition of IPTG to 0.4 mM in the medium for 12 h at 20°C, the E. coli cells were lysed by sonication on ice. After that, the cell lysate was centrifuged to remove cell debris, then the supernatants were recovered and the insoluble fraction was assumed to consist of inclusion bodies. W: whole cell lysate; S: supernatant; I: inclusion bodies.
Fig 5.
In vitro processing of the recombinant fusion protein ACP-GFP contained in E. coli cell lysate was performed by mixing it with the clarified cell lysate of E. coli over-expressing protease TEV (supernatant-TEV), and subsequently target protein GFP was purified of by one-step Ni-chelating chromatography.
The supernatant of the cell lysate of E. coli expressing fusion protein ACP-GFP was mixed with supernatant-TEV at a volumetric ration of 10:1 and incubated at 25°C. Aliquots were removed for SDS-PAGE analysis after 0 h, 2 h, 4 h, 6 h and 8 h. lane 1, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-GFP; lane 2, the supernatant of the cell lysate of E. coli expressing protease TEV by vector pET-28b-MBP-TEV-WHZ; lane 3, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-GFP treated by addition of the purified protease TEV; lane 4, target protein GFP purified by one-step Ni-chelating affinity chromatography.
Fig 6.
In vitro processing of the recombinant fusion protein ACP-G2-GlcK-Histag contained in E. coli cell lysate was performed by mixing it with the clarified cell lysate of E. coli over-expressing protease TEV (supernatant-TEV), and subsequently target protein GlcK was purified by one-step Ni-chelating chromatography.
The supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-GlcK-Histag was mixed with supernatant-TEV at a volumetric ration of 10:1, and incubated at 25°C. Aliquots were removed for SDS-PAGE analysis after 0 h, 2 h, 4 h, 6 h and 8 h. lane 1, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-GlcK-Histag; lane 2, the supernatant of the cell lysate of E. coli expressing protease TEV by vector pET-28b-MBP-TEV-WHZ; lane 3, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-GlcK-Histag treated by addition of the purified protease TEV; lane 4, target protein GlcK purified by one-step Ni-chelating affinity chromatography.
Fig 7.
In vitro processing of the recombinant fusion protein ACP-G2-Amy-Histag contained in E. coli cell lysate was performed by mixing it with the clarified cell lysate of E. coli over-expressing protease TEV (supernatant-TEV), and subsequently target protein Amy was purified by one-step Ni-chelating chromatography.
The supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-Amy-Histag was mixed with supernatant-TEV at a volumetric ration of 10:1 and incubated at 25°C. Aliquots were removed for SDS-PAGE analysis after 0 h, 2 h, 4 h, 6 h and 8 h. lane 1, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-Amy-Histag; lane 2, the supernatant of the cell lysate of E. coli expressing protease TEV by vector pET-28b-MBP-TEV-WHZ; lane 3, the supernatant of the cell lysate of E. coli expressing fusion protein ACP-G2-Amy-Histag treated by addition of the purified protease TEV; lane 4, target protein Amy purified by one-step Ni-chelating affinity chromatography.
Fig 8.
The protocol presented for purification of the target proteins, which need to be expressed in fusion form.
The passenger proteins that either accumulate dominantly in the soluble form after being cleaved off the fusion tags by the site-specific protease TEV co-expressed in vivo or are ready to form aggregates when processed intracellularly, can be convenient purified by one-step Ni-chelating affinity chromatography; RS: protease TEV recognition site.
Table 1.
Characterization of vectors constructed.