Thioredoxin Fusions Increase Folding of Single Chain Fv Antibodies in the Cytoplasm of Escherichia coli: Evidence that Chaperone Activity is the Prime Effect of Thioredoxin

Jurado, Paola; Lorenzo, Vı́ctor de; Fernández, Luis A.

doi:10.1016/j.jmb.2005.12.058

Cited by 79 publications

(69 citation statements)

References 49 publications

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“…In fact, the oxidation of cysteines in the E. coli cytoplasm is highly disfavored, due not only to the low redox potential but also to the absence of enzymes that catalyze protein thiol oxidation in the cytoplasm (4). In this context, it is generally accepted that the double mutant TrxB Ϫ Gor Ϫ strain (Origami), which has an intracellular environment shifted toward a more oxidative state, is the most useful cell factory for the production of proteins that contain disulfide bonds (22) and a good tool for understanding the role of disulfide bond formation in recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

Seras‐Franzoso

Affentranger

Ferrer-Navarro

et al. 2012

Appl Environ Microbiol

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ABSTRACTEscherichia coliβ-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version ofEscherichia coliβ-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.

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Section: Discussionmentioning

confidence: 99%

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

Seras‐Franzoso

Affentranger

Ferrer-Navarro

et al. 2012

Appl Environ Microbiol

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“…pl). The choice of thioredoxin as the fusion partner was based on the literature data indicating that the formation of disulfide was essential for many scFvs described (Jurado et al, 2006;Sonoda et al, 2010).…”

Section: Construction and Expression Of The Scfv -Mab6-9-1 Derivativementioning

confidence: 99%

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

Sawicka¹,

Siedlecki²,

Kalenik³

et al. 2016

Acta Biochim Pol

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Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.

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“…Other approaches to facilitate proper folding of scFv during expression include the co-expression of the scFv with chaperone proteins [159]. The fusion of the scFv construct to naturally occurring E. coli proteins (e.g., maltose-binding protein and thioredoxin) also resulted in an increase in properly folded, active scFv [160,161]. The successful production of a thioredoxin-fusion scFv in a co-expression system with chaperone protein, which resulted in increased specific expression of the fusion scFv, was reported recently [162].…”

Section: Innovative Approaches To Production Of Antibody Fragmentsmentioning

confidence: 99%

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

Cheng

Allen

2010

Expert Opinion on Drug Delivery

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Importance of the field-Targeted liposomal drugs represent the next evolution of liposomal drug delivery in cancer treatment. In various preclinical cancer models, antibody-targeted PEGylated liposomal drugs have demonstrated superior therapeutic effects over their non-targeted counterparts. Single chain Fv (scFv) has gained popularity in recent years as the targeting agent of choice over traditional targeting agents such as monoclonal antibodies (mAb) and antibody fragments (e.g., Fab′).Areas covered in this review-This review is focused mainly on advances in scFv-targeted liposomal drug delivery for the treatment of cancers, based on a survey of the recent literature, and on experiments done in a murine model of human B-lymphoma, using anti-CD19 targeted liposomes targeted with whole mAb, Fab′ fragments and scFv fragments.What the reader will gain-This review examines the recent advances in PEGylated immunoliposomal drug delivery, focusing on scFv fragments as targeting agents, in comparison with Fab′ and mAb.Take home message-For clinical development, scFv are potentially preferred targeting agents for PEGylated liposomes over mAb and Fab′, owing to factors such as decreased immunogenicity, and pharmacokinetics/biodistribution profiles that are similar to non-targeted PEGylated (Stealth ® ) liposomes.

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Thioredoxin Fusions Increase Folding of Single Chain Fv Antibodies in the Cytoplasm of Escherichia coli: Evidence that Chaperone Activity is the Prime Effect of Thioredoxin

Cited by 79 publications

References 49 publications

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

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