Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli

Sonoda, Hidenori; Kumada, Yoichi; Katsuda, Tomohisa; Yamaji, Hideki

doi:10.1016/j.jbiosc.2010.12.015

Cited by 36 publications

(38 citation statements)

References 24 publications

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“…Till today, a number of methods have been used to express the scFv antibody, including expression of affinity tag fusion (Esposito and Chatterjee, 2006), co-expression of molecular chaperones, and folding modulators (De Marco and De Marco, 2004; Sonoda et al, 2011), extracellular accumulation in a defined medium (Fu, 2010), refolding scFv using detergent and additive (Kudou et al, 2011) and expression in different host systems (Goulding and Perry, 2003). Amongst of these methods, expression of affinity tags fusion protein is the common method to improve the solubility of target proteins.…”

Section: Introductionmentioning

confidence: 99%

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Wang

Xiang

Feng

et al. 2013

Front. Cell. Infect. Microbiol.

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Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

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Section: Introductionmentioning

confidence: 99%

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Wang

Xiang

Feng

et al. 2013

Front. Cell. Infect. Microbiol.

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“…We thought it likely that some periplasmic factor must be assisting LacZ folding. Because previous studies have implicated the periplasmic chaperones skp and fkpA in the folding of heterologous proteins (23)(24)(25)(26)(27)(28)(29), we chose to see what effect disruption of these genes might have on the ␤-galactosidase activity of a strain expressing the malF-lacZ102 fusion in the absence of DsbA. While disruption of skp did not decrease ␤-galactosidase activity, we found that disruption of fkpA reduced it by a factor of 2.6 (Fig.…”

Section: Resultsmentioning

confidence: 92%

Folding LacZ in the Periplasm of Escherichia coli

et al. 2014

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b Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic ␤-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

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“…Over the past several decades, a variety of methods for producing a stable, soluble, and higher-affinity scFv antibody have been developed, including coexpression with molecular chaperons (3,31), expression in different host systems (32), genetic fusion to specific proteins (33), and GFP-based binders (29,30). Perhaps the most effectively used method is the use of a new type of antibody based on a GFP scaffold.…”

Section: Discussionmentioning

confidence: 99%

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Wang

Xiang

Zhang

et al. 2014

Appl Environ Microbiol

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Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

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Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli

Cited by 36 publications

References 24 publications

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

Folding LacZ in the Periplasm of Escherichia coli

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

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