Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm

Mahgoub, Elham O

doi:10.1007/s10528-012-9506-4

Cited by 7 publications

(7 citation statements)

References 30 publications

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“…Intracellular overexpression of antibody fragments often results in insoluble aggregates (inclusion bodies) and periplasmic expression generally suffers from low yields. The use of mutant strains that promote disulfide bond formation in the cytoplasm also represents for these antibody derivatives a successful and economic strategy to produce recombinant antibodies with protein productivities of up to 30 mg/L [49-51]. …”

Section: Discussionmentioning

confidence: 99%

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

et al. 2013

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BackgroundFor their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins.ResultsBy the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target.ConclusionsOur study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.

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Section: Discussionmentioning

confidence: 99%

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

et al. 2013

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“…It was reported that the optimization of cultivation conditions is useful to enhance soluble protein expression (Mahgoub, 2012). In this study, the effect of duration and FIGURE 2 -Optimization of anti-HER2 scFv expression condition in Origami (DE3).…”

Section: Discussionmentioning

confidence: 99%

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

Farshdari

Ahmadzadeh

Nematollahi

et al. 2020

Braz. J. Pharm. Sci.

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The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2 + tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemically synthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 °C. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 °C).

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“…The advantage using of scFv antibody in tumours labelling is clear, for example when scFv antibody is fused to technetium-99 m [26]. Technetium is commonly used since it has short half-life of six-hour.…”

Section: Scfv Antibodies In Tumours Labelling and Bio-therapeuticsmentioning

confidence: 99%

Single Chain Fragment Variables Antibody binding to EGF Receptor in the Surface of MCF7 Breast Cancer Cell Line: Application and Production Review

Mahgoub¹

2017

OJGen

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In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.

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Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm

Cited by 7 publications

References 30 publications

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

Single Chain Fragment Variables Antibody binding to EGF Receptor in the Surface of MCF7 Breast Cancer Cell Line: Application and Production Review

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