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

Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

doi:10.3389/fcimb.2013.00072

Cited by 49 publications

(46 citation statements)

References 42 publications

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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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Section: Resultsmentioning

confidence: 92%

Folding LacZ in the Periplasm of Escherichia coli

et al. 2014

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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%

“…T o date, antibodies are used for a very broad and still steadily expanding spectrum of applications, such as cancer therapy, disease diagnosis, and signal pathway investigation (1)(2)(3). Monoclonal antibodies (Abs) produced by hybridoma technology have been widely used to rapidly detect pathogens in food and raw seafood (4).…”

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confidence: 99%

“…The single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of heavy (V H ) and light (V L ) chains of immunoglobulin gene through a short polypeptide linker (3) and still retains the binding activity to target antigen (5,6). The smaller size of scFv fragment allows better tissue penetration, leading to improve tumor targeting and enhanced bloodbrain barrier permeability for the treatment of neurodegenerative diseases (7).…”

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confidence: 99%

“…In comparison to polyclonal or hybridoma antibodies, scFv antibody may be easily manipulated for improving specificity and affinity, thereby reducing the production cost (11,12). However, these applications of scFv were limited by drawbacks, such as the formation of an inclusion body, which often leads to low binding activity and an unstable structure and is cytotoxic to host cells (3). Hence, it is very important to develop a feasible approach for reducing these limitations.…”

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confidence: 99%

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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 variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

Koçer

Cox

DeLisa

et al. 2020

Biotechnology Progress

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Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

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

Cited by 49 publications

References 42 publications

Folding LacZ in the Periplasm of Escherichia coli

Folding LacZ in the Periplasm of Escherichia coli

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

Effects of variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

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