The ORF of TEV-protease was amplified from a cDNA clone and ligated into the BamHI and
EcoRI restriction sites of the pGEX-4T2 expression vector. The vector for the expression of
GST-tagged TEV protease (GST-TEV) was amplified in E. coli DH5α and after restriction tests transformed into E.coli BL21. Positive colonies were selected on ampicillin-LB-agar plates and grown as overnight cultures. As a test-expression, 20 ml LB were inoculated with GST-TEV overnight-cultures. The plain pGEX vector expressing GST was used as a control. Expression was induced with 100 µM IPTG at an optical density of OD600=0.6-0.7 and protein
samples were taken after 90 min and over night incubation at 37°C and analyzed by 12% SDS-PAGE. The 55 kDa band of overexpressed GST-TEV and the 26 kDa band of the GST- control could be easily detected (see Figure 3-34).
Figure 3-34 10% SDS-PAGE (Coomassie stain) of GST-TEV test expression. After over night (ON) expression the 55 kDa band of GST-TEV can be seen for both clones. Plain GST was expressed as a control.
As the expression in BL21 cells was rather weak, the GST-TEV expression vector was transformed into E. coli BL21 DE3 pLysS. The expression experiment was repeated with two clones of each strain in 20 ml LB as described above. After expression over night, the cells were harvested by centrifugation and purified according to the pGEX-protocol (Amersham Bioscience). SDS-PAGE of the pellets and supernatants obtained by centrifugation of the homogenized lysates revealed that the protein exclusively resided in the pellet. No difference could be determined between the expression levels in E.coli BL21 and BL21 DE3 pLysS.
Figure 3-35 10% SDS-PAGE of GST-TEV lysates. After cell lysis according to the pGEX-protocol the recombinant protein resides in the pellet (P). Expression in BL21 or BL21 DE3 pLysS and at 37°C (a) leads to no significant changes in the amount of soluble protein. Only poor yields of recombinant protein are obtained by overnight expression at 17°C (b). (ON = overnight culture, P = pellet, S = supernatant, the numbers refer to the clone).
As the amount of soluble protein may depend on the expression temperature, the experiment was repeated with an incubation temperature of 17°C. However, overnight expression at these temperatures yielded very low levels of recombinant protein, so that no detectable amounts ob soluble GST-TEV were obtained after cell lysis.
Apparently, GST-TEV forms inclusion bodies. The two equally sized protein domains might interfere with each others folding process leading to the precipitation of the misfolded protein. To obtain soluble GST-TEV protease from inclusion bodies, a protocol for the purification of His6-TEV protease is applied with some modifications (Lucast et al. 2001).
The cell pellets were resuspended in lysis buffer supplemented with lysozyme and DNase. After 1h of incubation on ice, the cells were homogenized and the insoluble components pelleted by centrifugation. SDS-PAGE from pellets and lysates revealed that the major proportion of the overexpressed protein still resided in the pellet (data not shown).
Therefore, the pellet containing the insoluble protein was purified under denaturing conditions as described previously (Lucast et al. 2001). For affinity binding to glutathione- sepharose, the protein needs to be refolded. Therefore, the denatured protein solution was dialyzed against TEV-refolding buffer and pGEX-Lysis buffer, respectively. In both buffers a large fraction of the protein precipitated. The precipitate was removed by centrifugation and the supernantant was incubated with glutathione sepharose for 2h at room temperature to recover any fusion protein with a properly refolded GST-fusion tag. SDS-PAGE of the supernatants and eluates indicated that the recombinant protein was dissolved in the denaturing supernatant and in the dialysis sample albeit a major proportion of protein was lost due to precipitation. However, only very small amounts of the protein could be eluted from the resin (see Figure 3-36). GST-TEV bands in the eluates from the BL21 DE3 pLysS strain originate from a protein precipitate that occurred during the incubation with glutathione sepharose, that could not be fully removed from the resin by repeated washing.
Figure 3-36 Inclusion body preparation of GST-TEV expressed in BL21 and BL21 DE3 pLysS. The recombinant protein from the overnight culture (ON) forms inclusion bodies that are found in the insoluble pellet (P). Treatment of the pellet with denaturing buffer redissolves GST-TEV that is found in the dialyzed supernatant but does not bind to glutathione sepharose as shown by the eluates (E1-3) and the supernatant decanted from the resin (GS). Note the thin GST band that is copurified at ~26 kDa (12% SDS-PAGE, Coomassie Stain).
As the pH optimum for refolding is 7.0 for GST (Amersham Bioscience) and 8.5 for TEV- protease, the pH of the refolding buffer was adjusted to values between 7.5 and 8.5. The dialyzed protein solutions were incubated for 2h with glutathione-sepharose beads. During the incubation the protein precipitated, and no significant amounts of protein could be detected in the eluates with Bradford reagent or by 10% SDS-PAGE (see Figure 3-37). Photometric quantitation of the eluates against a BSA standard indicated that the protein yield was below 1 mg/l for both samples.
Figure 3-37 10% SDS-PAGE of GST-TEV refolding and purification experiments at different pH (Coomassie Stain). Dialysis to refolding buffer at pH 7.4 and 8.2 does not yield properly refolded protein, so that no GST-TEV can be bound by glutathione-sepharose and no protein can be seen in the eluates. (ON = overnight expression culture, S = supernatant from cell lysis, DS = denaturing supernatant, GS = supernatant after incubation with glutathione sepharose, P = pellet from cells lysis , E = eluates).
The precipitated protein could neither be redissolved in larger volumes of refolding or pGEX buffer nor by the addition of DTT to a concentration of 2 mM. Also, sonification with a cup horn could not resdissolve the protein.