Bax is a pro-apoptotic protein which is also toxic to E. coli (40). This toxicity limits the expression of Bax in E. coli. The protocol developed in our laboratory takes advantage of the leakiness of the trc promoter within pGEX plasmids to achieve a slow gradual induction, using low concentrations of IPTG and modifications of sugar levels in media. Also, the presence of a GST tag at the N-terminus of Bax can reduce the toxicity of the recombinant protein in bacteria. We have not successfully produced large amounts of His6-Bax.
We have observed that, after cleavage and removal of GST, the recombinant Bax protein will non-specifically stick to GST resin unless «0.1%(v/v) non-ionic detergent is added. The concentration of purified Bax is limited to =0.2 mg per ml in the absence of detergent.
About half of the expressed GST-Bax protein is found in the insoluble pellet, probably associated with cell membrane debris. Non-ionic detergent does not increase the solubility of this pool of Bax. Bax protein found in the insoluble pellet, however, can be recovered through solubilization and refolding. The washed pellet is solubilized in 6 M GuHCl, 50 mM phosphate buffer, pH 7.0, 0.1% 2-mercaptoethonal. The supernatant is then diluted 30fold into ice-cold refolding buffer (50 mM phosphate-buffer, pH 7.0, 0.1% 2-mercaptoethonal, 150 mM NaCI, 0.1% Tween-20, lm EDTA) with vigorous stirring, then placed on ice for 16 h. After clarification by centrifugation, the diluted refolding mixture is mixed with GST-resin. This rapid dilution of GuHCl appears to be preferable to dialysis-based approaches to refolding, since when solubilized GST-Bax in GuHCl is dialysed against refolding buffer, most of GST-Bax protein appears as a high molecular weight aggregate.
A problem associated with the purification of Bax is that Bax can become cleaved during proteolytic removal of GST tag. Initially, we attempted an approach where GST-Bax was eluted and concentrated, then digested with thrombin. However, we found that most Bax is degraded under these conditions. Thus, digestion is performed directly on GST beads at 4°C for 24 h. Under these conditions, about 20% of Bax still becomes truncated near its N-terminus. If a homogeneous N-truncated Bax preparation is desirable, the eluted Bax protein from GST beads can be kept at 4°C for 1 week, which typically results in complete conversion of the Bax protein.
When chromatographed on Mono Q, Bax co-elutes at 350 mM NaCl in a broad peak. Although this ion-exchange step does not separate these proteins, it does separate Bax from contaminating thrombin and residual GST protein, and it can be used to remove detergents which have been employed in previous steps.
Purified GST-Bax, Bax, and truncated Bax all elute as high molecular weight complexes in gel filtration columns, migrating at =130-280 kDa. These complexes can withstand up to 4 M urea and up to 1 M NaSCN, as well as alkaline solutions to pH 9.5. In this regard, it has been reported that Bax may form heptamers (41). At low pH, Bax precipitates out of solution unless diluted into detergent-containing solutions or provided with membranes for insertion.
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