Oxidative refolding

The inherent drawbacks of the oxidative refolding approach for synthetic polypeptides containing multiple cysteine residues is the individual behavior of each peptide that derives from the encoded sequence, more or less pronounced structural information which prevents general procedures to be elaborated and proposed. Nevertheless, this synthetic approach remains attractive because of its simplicity compared to the synthetic strategies for re-gioselective disulfide bond formation (Section 6.1.1-6.1.4), and it is certainly indispensable if the number of cysteine residues exceeds the presently available chemistry for site-directed cysteine pairings. 

Oxidative Refolding of Peptides with Two Intramolecular Disulfide Bonds... 

Of this family of peptides containing two intramolecular disulfide bonds the most studied in terms of oxidative refolding are a-conotoxins with two adjacent cysteine residues, i.e. with m = 0, the bee venom toxins, for example apamin and mast cell degranulating peptide, and snake venom toxins, exemplified by sarafotoxins, and endothelins, mammalian peptide hormones with the characteristic Cys-(Xaa)rCys/Cys-(Xaa)3-Cys motif (Scheme 2). With m = 0 or 1 all these peptides are expected to show a weak tendency to form the isomer 3 with a disulfide bond between two proximal cysteine residues. This was fully confirmed by oxidative refolding experiments. 

All the disulfide-folding pathways have the common feature that disulfide formation is initially random and not energetically favored due to the unfolded state of the reduced polypeptide chain. Disulfide formation becomes less random as certain disulfides are favored energetically as a result of the acquisition of nonrandom local conformations. In turn, the disulfides stabilize such conformations, so that disulfide formation and folding become cooperative. Thereby the sequence encoded structural information may fully suffice to direct the correct oxidative refolding as shown for co-conotoxin GVIA (9) (Section 6.1.5.2.1) which assumes the native-type cystine connectivities directly even in absence of redox reagents and... 

Oxidative Refolding of Double-Stranded Cystine Peptides... 

Oxidative Refolding of Mixed Selenocysteine/Cysteine Peptides... 

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).

Fig. 8. Acceleration of the oxidative refolding of RNase T1 by PPI and PDI. The increase in fluorescence at 320 nm is shown as a function of the time of reoxidation. The final conditions were 2.5 fiM RNase T1 in 0.1 Af Tris-HCl, 0.2 M GdmCl, 2 mM EDTA, 3 mAf glycine, 0.4 mAf oxidized glutathione, and 4 mAf reduced glutathione at pH 7.8 and 25°C. Reoxidation ( ) in the absence of PPI and PDI, (O) in the presence of 1.4 tiM PPI, (A) in the presence of 1.6 fiM PDI, and (A) in the presence of both 1.6 fiM PDI and 1.4 /uAf PPI. In all experiments more than 90% of the observed kinetics were well approximated by single first-order processes, as indicated by the continuous lines. The respective time constants (t) are ( ) t = 4300 sec, (O) r = 2270 sec, (A) t = 1500 sec, (A) T = 650 sec. In all cases the initial fluorescence signal was about 10% of the final emission of the native protein. From Schonbrunner and Schmid (1992).

Takesada et al. (1973) used CD in a study of the unfolded proteins and concluded that the two differ, with much structure remaining for a-lactalbumin that was not present for lysozyme (see also Section 1X,E). CD has been used also in the search for conformational intermediates, in a comparative study of the oxidative refolding of lysozyme and a-lactalbumin (Kuwajima et al., 1985). This topic is dealt with in Section IX,E. 

Oxidative Refolding of Recombinant Human Glia Maturation Factor Beta... 

ZAHEER LIM Oxidative Refolding of Recombinant Maturation Factor 81... 

Fig. 2. Reverse-phase HPLC profile of r-hGMF-beta. (A) Pure r-hGMF-beta without treatment. (B) After treatment with 10 mM DTT in presence of 6M guanidine hydrochloride. Note identical retention time in each case. (C D) Time course of oxidative refolding of r-hGMF-beta and separation of isoforms by reverse-phase HPLC. Pure r-hGMF-beta was dissolved in O.IM sodium phosphate and incubated at room temperature with reduced and oxidized glutathione in the presence of guanidine hydrochloride, as described in the text, for 4 h (C) and 8 h (D). (Adapted from ref. 17)...

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