Acceleration of disulfide‐coupled protein folding using glutathione derivatives

Abstract

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives – glutamyl‐cysteinyl‐arginine (ECR) and arginyl‐cysteinyl‐glycine (RCG) – and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (KRCG = 3.69 × 10−3 s−1) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide‐exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.

Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase

“…The digested peptide fragments were analyzed by HPLC (GL Science) equipped with a COSMOSIL 5C 18 -AR-II column (Nacalai Tesque) pre-equilibrated with 5% acetonitrile in 0.05% TFA and eluted with a linear acetonitrile gradient ranging from 5% to 65% at a rate of 1%/min, with detection at an absorbance of 220 nm. The molecular masses of separated peptides were determined by MALDI-TOF MS (Bruker Daltonics) (36,37). Synthetic peptides were prepared by the Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase method, modified with AMS, and analyzed by HPLC and MALDI-TOF MS as described previously (5,34,37).…”

Expression, Solubilization and Refolding Of Antigen-Binding Fragments Of Antibodies Fused With Leucine Zipper In Escherichia Coli

“…Second, refolding of the solubilized protein by the step-wise dialysis and the incubation with GSSG and L-arginine at the appropriate stage (1 M and 0.5 M of urea). The GSSG promoted formation of disulfide bonds in proteins [8] and Larginine suppressed aggregation and prevented incorrect of intramolecular disulfide bonds [9,10]. In summary, refolding procedures in this study is easy, inexpensive, time-saving and can be utilized for a high yield recovery of Fab inclusion bodies produced in E. coli.…”

New Methods for Chemical Protein Synthesis

“…The use of thiols containing positively charged groups has also recently been investigated by a different group working on analogs of glutathione. The Hidaka group has done extensive work with the tripeptide ArgCys-Gly (RCG) and its use as a redox buffer [184,185]. They have shown that an RCG-based buffer system resulted in a significantly higher folding recovery of lysozyme as compared to glutathione.…”