Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Wang, Ming-Rong; Zhang, Yongxia; Du, TianFei; Dai, Yue; He, Yi; Yu, Xiaoyan; Cong, Cong; He, Mingyue

doi:10.4161/mabs.3.5.16087

Cited by 3 publications

(6 citation statements)

References 14 publications

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“…With the His 6 tag at the C terminus, the captured scFv was detected by a HRP-linked anti-His 6 antibody. The relative soluble level of scFv was then measured through a side-by-side comparison with the original clone, which generally yielded the scFv at the level of ϳ1 mg/liter (17). Our results revealed a huge difference in protein solubility among the variants, ranging from completely insoluble aggregates to a soluble yield of ϳ2.5 mg/liter ( Fig.…”

Section: Design and Construction Of Synonymous Codon Libraries-mentioning

confidence: 98%

“…Library Design and Construction-A human anti-IgE scFv composed of 258 amino acids, including a flexible linker (GGGS) 4 and a C-terminal His 6 tag, was used as the template (17) to construct the synonymous codon libraries. The E. coli B Codon Usage Database was chosen as a guide to design the codon mix that contained high, medium, and low usage frequencies (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…The total scFv expression was obtained by adding the sarkosyl-solubilized scFv and Bugbuster-extracted scFv together. To purify soluble scFv for ELISA analysis, nickel-agarose (Novagen) was used as described (17). For UV CD analysis, scFv was first purified by protein L-agarose (Captol L, GE Healthcare) following the manufacturer's instructions and then by nickel-agarose purification as described (17).…”

Section: Methodsmentioning

confidence: 99%

“…This showed that the IgE-binding activity differed from no binding at all to an affinity greater than the original scFv (10 Ϫ8 M) ( Fig. 2 and supplemental Table S1) (17). To further characterize the binding activity, four variants, together with the original scFv, were expressed and purified using a His-tagged affinity column.…”

Section: E Coli Expression and Screening For Altered Functionality-mentioning

confidence: 99%

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Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Wang

Cai

et al. 2013

Journal of Biological Chemistry

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Background: Synonymous codon usage affects protein properties in a given organism. Results: A total of 342 antibody codon variants were identified, differing significantly in solubility and functionality while retaining the identical original amino acid sequence. Conclusion: Genetic codes control protein synthesis and folding. "Codon-preferred" DNA template(s) can be generated by functional screening. Significance: Protein properties can be considerably altered by synonymous codons without substituting amino acids.

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Section: Design and Construction Of Synonymous Codon Libraries-mentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: E Coli Expression and Screening For Altered Functionality-mentioning

confidence: 99%

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Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Wang

Cai

et al. 2013

Journal of Biological Chemistry

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“…scFvs were expressed in E. coli as described [11]. A (His) 6 tag was introduced at the C-terminus of scFvs for the purpose of detection and purification.…”

Section: Scfv Expression and Screening For Specific Bindersmentioning

confidence: 99%

Cell‐free ribosome display and selection of antibodies on arrayed antigens

Cong¹,

Yu²,

He³

et al. 2016

Proteomics

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In vitro display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free ribosome display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.

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Generation of scFv specific to human VEGFR-3 from the neutralizing mAb BDD073

Chen

Wang

Gao³

et al. 2014

Protein Engineering Design and Selection

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In our previous study, we have produced a neutralizing mAb of vascular endothelial growth factor receptor 3 (VEGFR-3), specifically BDD073, which could inhibit angiogenesis in the CAM model. However, the clinical application of BDD073 is restricted due to its mouse origin, which might cause human anti-mouse antibody reactions. Herein, we generated functional recombinant single-chain variable fragments (scFv) from mAb BDD073 producing mouse hybridoma cells. The scFv gene containing variable regions of heavy and light chains of BDD073 was cloned into an expression vector with trx tag and expressed in Escherichia coli BL21 (DE3). The recombinant scFv was purified and refolded with Ni-NTA agarose metal affinity column. The bacterially expressed scFv showed moderate potency and specificity to the human VEGFR-3. It may serve as a potential candidate of anti-VEGFR3 treatment for biotechnological and therapeutic applications.

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Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Cited by 3 publications

References 14 publications

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Cell‐free ribosome display and selection of antibodies on arrayed antigens

Generation of scFv specific to human VEGFR-3 from the neutralizing mAb BDD073

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