Section 3.1.5.3. Purifying and refolding denatured proteins

As we have already discussed, expressing high levels of recombinant prokaryotic or eukaryotic proteins in E. coli can lead to the production of improperly folded material that aggregates to form insoluble inclusion bodies (Marston, 1986; Krueger et al., 1989; Mitraki & King, 1989; Hockney, 1994). Inclusion bodies can usually be recovered relatively easily, following lysis of cells by low-speed centrifugation (5 min at 12 000 g); inclusion bodies are larger than most macromolecular structures found in E. coli and denser than E. coli membranes. Care should be taken to achieve complete lysis, since an intact bacterial cell that remains after lysis will co-sediment with the inclusion bodies. In most (but not all) cases, the inclusion bodies contain the desired recombinant protein in relatively pure form. In such cases, the problem lies not with the purification of the protein, but in finding a proper way to refold it.

Various general procedures for refolding proteins from inclusion bodies have been described (Fischer et al., 1993; Werner et al., 1994; Hofmann et al., 1995; Guise et al., 1996; De Bernardez Clark, 1998), and the literature is filled with examples of specific protocols. The insoluble inclusion bodies are usually solubilized in a powerful chaotropic agent like guanidine hydrochloride or urea. In general, detergents are not recommended. The denaturant is sequentially removed by dilution, dialysis or filtration. Both rapid dilution and slow removal of the denaturant have been used successfully. In most refolding protocols, relatively dilute solutions of the protein are used to avoid protein–protein interactions, and, if necessary, glutathione or some other thiol reagent is included in the buffer to accelerate correct pairing of disulfides. After a refolding procedure, the properly folded soluble protein must be separated from the fraction that did not fold appropriately. Improperly refolded proteins are relatively insoluble and can usually be removed by centrifugation. It is sometimes profitable to try to refold the recovered insoluble material a second time.

Once soluble protein has been obtained, conventional purification procedures may be employed. It should be noted that recovery of soluble protein is not necessarily an indication that the protein exists in a native state. Quantitative assays of protein activity should be used to characterize the protein, if such assays exist. Alternatively, the behaviour of the refolded protein should be critically assessed during subsequent purification steps; an improperly folded protein will be prone to aggregation, will generally give broad and/or trailing peaks during column chromatography and will migrate faster than expected during size-exclusion chromatography. Some proteins are more amenable to refolding than others. As has already been pointed out, if a protein has a complex array of disulfide bonds, it is usually more difficult to refold than a protein without disulfide bonds. Greater success in refolding is generally obtained with proteins composed of single domains than with multidomain proteins.