The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential

Wetlaufer, Donald B.; Branca, Phillip A.; Chen, Guo-Xian

doi:10.1093/protein/1.2.141

Cited by 67 publications

(33 citation statements)

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“…The pioneering work of Saxena and Wetlaufer (28) and Wetlaufer et al (29) was adapted to make an effective redox buffer composed of 3 mM cysteine, 0.3 mM cystine, 1 mM EDTA to facilitate the formation of the three native disulfide bonds. The RBP concentration during refolding was optimized to be 0.25 mg/mL, giving a satisfactory refolding yield of 50ϳ60% in the presence of a 10-fold excess of vitamin A.…”

Section: Resultsmentioning

confidence: 99%

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Xie

Lashuel

Miroy

et al. 1998

Protein Expression and Purification

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Section: Resultsmentioning

confidence: 99%

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Xie

Lashuel

Miroy

et al. 1998

Protein Expression and Purification

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“…Generally, proteins assembled in the ER are rich in disulfide bonds that help to promote efficient folding and oligomerization of nascent polypeptide chains (9). While disulfide bond formation occurs spontaneously and independently of enzymatic processes, to ensure correct intramolecular disulfide bonding is achieved, the ER contains chaperones and oxidoreductases to ultimately produce a properly folded protein (20,25,32,33,41). The activities of resident ER folding enzymes are highly dependent on the reduction/oxidation (redox) environment, and even small perturbations in redox status greatly affects enzymatic activity and, as a result, protein folding kinetics (27,28,30,31).…”

Section: Introductionmentioning

confidence: 99%

Assessment of Endoplasmic Reticulum Glutathione Redox Status Is Confounded by ExtensiveEx VivoOxidation

Dixon

Heath

Kim³

et al. 2008

Antioxidants & Redox Signaling

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Glutathione (GSH) and glutathione disulfide (GSSG) form the principal thiol redox couple in the endoplasmic reticulum (ER); however, few studies have attempted to quantify GSH redox status in this organelle. To address this gap, GSH and GSSG levels and the extent of protein glutathionylation were analyzed in rat liver microsomes. Because of the likelihood of artifactual GSH oxidation during the lengthy microsomal isolation procedure, iodoacetic acid (IAA) was used to preserve the physiological thiol redox state. Non-IAA-treated microsomes exhibited a GSH:GSSG ratio between 0.7:1 to 1.2:1 compared to IAA-treated microsomes that yielded a GSH:GSSG redox ratio between 4.7:1 and 5.5:1. The majority of artifactual oxidation occurred within the first 2 h of isolation. Thus, the ER GSH redox ratio is subject to extensive ex vivo oxidation and when controlled, the microsomal GSH redox state is significantly higher than previously believed. Moreover, in vitro studies showed that PDI reductase activity was markedly increased at this higher thiol redox ratio versus previously reported GSH:GSSG ratios for the ER. Lastly, we show by both HPLC and Western blot analysis that ER proteins are highly resistant to glutathionylation. Together, these results may necessitate a re-evaluation of GSH and its role in ER function.

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“…The reoxidation of thiol groups during dialysis was probably effected by 2-hydroxyethyldithiol, a disulphide-bridged dimer generated by aerobic oxidation of 2-mercaptoethanol [7]. Reoxidation proceeds via a mixed disulphide intermediate formed by the reaction of ionised thiol groups with 2-hydroxyethyldithiol [27].…”

Section: Discussionmentioning

confidence: 99%

Refolding human serum albumin at relatively high protein concentration

Burton¹,

Quirk²,

Wood³

1989

European Journal of Biochemistry

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Thc conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2-mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4-8 M urea and 14 mM 2-mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 pM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg . ml-' whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding.A common approach to solving the enigma of protein folding has been to observe the refolding of reversibly denatured proteins in vitro. The intrinsic ability of large, disulphide-bonded proteins to refold correctly from a reduced and denatured state was demonstrated by Anfinsen and coworkers from their studies on ribonuclease [l, 21. Since then a number of proteins have been refolded to their original native conformations [3 -61. Traditional protocols for refolding reduced and denatured proteins involve the removal of the chaotrope and thiol reductant by dilution or dialysis in an oxidative environment. To combat the frequently encountered problems of protein aggregation and incorrectly formed disulphide bonds, the denatured protein is commonly diluted to micromolar concentration and refolded in the presence of disulphide/thiol containing buffers [4, 71 or disulphide exchange enzymes [8, 91. Interest in protein refolding has intensified in recent years with the advent of recombinant DNA technology. Heterologous proteins expressed in microorganisms are frcquently insoluble within the cell and their recovery in a soluble form often necessitates renaturation of a chaotrope-reductant extract. Large-scale protein refolding places high demands on the renaturation protocol, since long refolding times, large volumes of dilute protein solution and poor recoveries of native material are unacceptable in a production process. Thus a requirement exists for highly efficient protein refolding regimes.Serum albumin provides an interesting model for protein refolding studies since, in order to regenerate the native protein 17, disulphide bonds must be correctly reformed [3]. Human serum albumin (HSA) is also a valued therapeutic product which has been expressed as an intracellular recombinant protein in a number of organisms [lo-121. In the majority of these examples the recombinant HSA forms C,'itrrespontf~~nce to A. V. Quirk,

show abstract

The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential

Cited by 67 publications

References 0 publications

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Recombinant Human Retinol-Binding Protein Refolding, Native Disulfide Formation, and Characterization

Assessment of Endoplasmic Reticulum Glutathione Redox Status Is Confounded by ExtensiveEx VivoOxidation

Refolding human serum albumin at relatively high protein concentration

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