Removal of the conserved disulfide bridges from the scfv fragment of an antibody: effects on folding kinetics and aggregation

Ramm, Kathrin; Gehrig, Peter; Plückthun, Andreas

doi:10.1006/jmbi.1999.2854

Cited by 51 publications

(54 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is also interesting to note that mutation of the cysteine residues did not significantly alter antibody expression (on the yeast surface in this case; Fig. 1 A), in contrast to other published reports (15,36).…”

Section: Discussioncontrasting

confidence: 56%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

145

View full text Add to dashboard Cite

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets

show abstract

Section: Discussioncontrasting

confidence: 56%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

145

View full text Add to dashboard Cite

show abstract

“…The importance of the disulfide linkage in the immunoglobulin fold has been demonstrated in a large number of studies (2,10,19), and recent studies (31-33) on disulfide-free antibody suggest that the stabilization of the disulfide-free antibodies requires amino acid exchange to compensate for the loss of folding stability upon cleavage of the disulfide linkage, although a functional disulfide-free scFv can be obtained. A mutation study on the scFv of the antibody hu4D5-8 against the extracellular domain of human epidermal growth factor receptor-2 indicates that the wild-type 4D5 scFv with a disulfide linkage in each of V H and V L domains has a higher midpoint of denaturation in the equilibrium transition than the variants lacking one or two disulfide linkages (10), which means that the 4D5 scFv with the native disulfide linkages is folded at a higher GdnHCl concentration than the variants.…”

Section: The Formation Of Disulfide Linkages In the Stepwise Dialysismentioning

confidence: 99%

“…A mutation study on the scFv of the antibody hu4D5-8 against the extracellular domain of human epidermal growth factor receptor-2 indicates that the wild-type 4D5 scFv with a disulfide linkage in each of V H and V L domains has a higher midpoint of denaturation in the equilibrium transition than the variants lacking one or two disulfide linkages (10), which means that the 4D5 scFv with the native disulfide linkages is folded at a higher GdnHCl concentration than the variants.…”

Section: The Formation Of Disulfide Linkages In the Stepwise Dialysismentioning

confidence: 99%

“…For the variable fragment in antibody molecules (Fv), which is composed of heavy and light chain domains (V H and V L , respectively), the immunoglobulin fold is known to be partially and irreversibly denatured to an aggregate under reducing conditions (2); furthermore, the instability of the Fv that is missing its disulfide linkages has been proved by natural antibodies missing a disulfide linkage (8) and replacement of the essential cysteinyl residue by serine residue (9). Recently, the folding kinetics and aggregation behavior of the single-chain Fv fragment (scFv) after removal of the disulfide linkages have been analyzed by fluorescence spectroscopy and a combination of mass spectrometry and an H/D exchange technique (10,11). In the folding of the disulfide-containing scFv in vitro, the presence of the disulfide linkage accelerates the independent folding of the V H and V L domains, and then the domains are associated with each other to form the correct quaternary structure.…”

mentioning

confidence: 99%

“…In the folding of the disulfide-containing scFv in vitro, the presence of the disulfide linkage accelerates the independent folding of the V H and V L domains, and then the domains are associated with each other to form the correct quaternary structure. For the scFv with a domain containing no cysteinyl residues, the disulfide-free domain refolds more slowly than the disulfide-containing domain in the same scFv, and refolding requires an interface on the domain with a native disulfide linkage to form a functional structure (10). It has also been noted that when V H folds prior to interaction with the V L domain, the native structure results, whereas when the stable V L domain is formed before V H , aggregate formation is likely (10).…”

mentioning

confidence: 99%

See 2 more Smart Citations

How Additives Influence the Refolding of Immunoglobulin-folded Proteins in a Stepwise Dialysis System

Umetsu¹,

Tsumoto²,

Hara³

et al. 2003

Journal of Biological Chemistry

162

100

View full text Add to dashboard Cite

The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and L-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 M GdnHCl to 1 M GdnHCl, and 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid dipotassium salt binding analysis indicated that the addition of L-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether L-arginine was present. Spontaneous folding at the 1 M GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of Larginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of L-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether L-arginine was present. Controlled coupling of the effects of GSSG and L-arginine led to the complete refolding of scFv in the stepwise dialysis.The internal disulfide linkage in the immunoglobulin fold is of particular importance for most proteins in the immunoglobulin superfamily because this linkage has a critical influence on the stability of these proteins (1, 2). The linkage, which is highly conserved, connects two ␤-sheets in a sandwich structure (3-6). In the case of antibody molecules, Goto and Hamaguchi (1, 7) were the first to analyze intrachain disulfide bond formation in the constant fragments of the immunoglobulin light chain (C L ), 1 demonstrating the contribution of the disulfide linkage to the stabilization and folding of the C L domain. For the variable fragment in antibody molecules (Fv), which is composed of heavy and light chain domains (V H and V L , respectively), the immunoglobulin fold is known to be partially and irreversibly denatured to an aggregate under reducing conditions (2); furthermore, the instability of the Fv that is missing its disulfide linkages has been proved by natural a...

show abstract

Assessment of the effect of triton X‐114 on the physicochemical properties of an antibody fragment

Malpiedi

Nerli

Abdalla

et al. 2014

Biotechnology Progress

View full text Add to dashboard Cite

The effect of Triton X-114 on the physicochemical properties of a single-chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid-liquid extraction procedures.

show abstract

Removal of the conserved disulfide bridges from the scfv fragment of an antibody: effects on folding kinetics and aggregation

Cited by 51 publications

References 29 publications

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

How Additives Influence the Refolding of Immunoglobulin-folded Proteins in a Stepwise Dialysis System

Assessment of the effect of triton X‐114 on the physicochemical properties of an antibody fragment

Contact Info

Product

Resources

About