Refolded proteins from aggregates

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

Comparative sequence analysis of even well described inter-protein interactions (i.e., from X-ray structure of multi-domain or subunit proteins) has only recently been possible initial results have however shown some common features of quaternary interactions. There is hope that interactions in naturally selfassociating proteins will be similar to those that lead to irreversible interactions in refolded proteins, and that one can then use this knowledge to prevent precipitation. For example, fibrin clots can be dissolved by adding the tetra-peptide Gly-Pro-Arg-Pro, the amino acids at the N-termini of fibrinogen molecules after thrombin cleavage. A related peptide, Arg-Gly-Asp-Ser, from the carboxy- terminus of fibrinogen, inhibits platelet aggregation. ... 

Although STET appears to be the best extraction buffer for purification of the CK pellet, the specific activity of CK refolded from this material was still considerably lower than that for the tissue purified control, indicating the need for further purification. The use of anion exchange FPLC in 8 M urea provided this additional level of purification, and samples refolded from this material have specific activities that are comparable to those of CK refolded from tissue-purified controls. We conclude that purification in the denatured state by FPLC apparently removes the contaminating protease(s) and other proteins from the aggregate even more efficiently than either STCT or octyl glucoside extraction. 

This renaturation procedure may find wider application to the commercial-scale renaturation of other recombinant proteins from bacterial inclusion bodies. The only proviso appears to be the existence of one or possibly more partially folded states at intermediate denaturant concentrations. Structural homologs of BST such as porcine somatotropin (16) and bovine prolactin are candidates for this approach to refolding. We are currently developing a general model for the prediction of optimum denaturant concentration for protein refolding via a consideration of the tradeoffs between aggregation and unfolding phenomena. 

St. John RJ, Carpenter J, Randolph T. High pressure fosters protein refolding from aggregates at high concentrations. Proc Natl Acad Sci USA 1999 96 13,029-13,033. 

It is not clear whether any of the Hsp70/40 chaperones also act downstream of Hspl04 to help refold proteins extracted from prion aggregates. Such a function appears very likely, but not critical, for prion propagation. 

This assists the refolding by reducing intermolecular aggregation. The refolding reaction is completed within the pores and there is minimal likelihood of protein aggregation. The protein is finally eluted from the column in its active native form. The size-exclusion characteristic of this process ensures that any aggregates that may have formed are removed from the column first, and the chaotropic species, which have low molecular weights in comparison to the protein are eluted after the refolded protein. 

Recently, SPs were used successfully for improving the refolding of recombinant proteins expressed in bacterial hosts, in the form of inactive and insoluble aggregates known as inclusion bodies (Gautam et al, 2012). From the biotechnological perspective, the recovery of an active protein from inclusion bodies is achieved in two steps ... 

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