High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Lehmann, Katrin; Hoffmann, Silke; Neudecker, Philipp; Suhr, Martin; Becker, Wolf‐Meinhard; Rösch, Paul

doi:10.1016/s1046-5928(03)00190-6

Cited by 54 publications

(41 citation statements)

References 30 publications

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“…In recent years, purified and/or recombinant allergen compounds have been used to determine the allergen reactivity of allergic patients [59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95]. The availability of such biochemically defined allergens has led to the development of specific, component-resolved diagnostic tests [59].…”

Section: Cd203c As a Diagnostic Tool In Allergy: Comparison With Cd63mentioning

confidence: 99%

The Basophil-Specific Ectoenzyme E-NPP3 (CD203c) as a Marker for Cell Activation and Allergy Diagnosis

Bühring

Streble

Valent

2004

Int Arch Allergy Immunol

215

199

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Basophils are effector cells of allergic reactions. These cells produce and store a number of vasoactive and immunomodulatory mediators. During an allergic reaction, basophils can release their mediator substances into the extracellular space and thus contribute to the clinical picture and symptoms in allergy. The phenotypic hallmark of basophils is expression of high-affinity IgE receptors (FcΕRI) on their cell surface together with expression of the activation-linked molecule CD203c. This ectoenzyme is located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the FcΕRI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release. CD203c is therefore a promising target molecule for a flow cytometry-based test to analyze sensitized individuals and patients with type I allergy. In the present article, we review the current knowledge of CD203c with special regard to its tissue distribution and regulation in basophil activation. In addition, we discuss the application of CD203c in allergy diagnosis.

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Section: Cd203c As a Diagnostic Tool In Allergy: Comparison With Cd63mentioning

confidence: 99%

The Basophil-Specific Ectoenzyme E-NPP3 (CD203c) as a Marker for Cell Activation and Allergy Diagnosis

Bühring

Streble

Valent

2004

Int Arch Allergy Immunol

215

199

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“…The recombinant protein was active, but the authors did not directly demonstrate proper disulfide bond formation (29). Ara h 2, the major peanut allergen, was expressed in E. coli Origami, and all of the cysteines were oxidized, indicating that they were all involved in disulfide bridges (37). DvnRV41 possesses four oxidized cysteine residues, as shown by mass spectrometry analysis.…”

Section: Discussionmentioning

confidence: 98%

“…According to Bessette et al (5), a higher yield of oxidized proteins can be obtained in the cytoplasm of the TRXB gor double mutant (gor encodes glutathione reductase). The expression of proteins by the pET-32b expression vector in E. coli Origami (DE3) has been successfully achieved for plant, bacterial, and human proteins (3,19,20,29,37). However, to our knowledge, no peptides containing two disulfide bridges have been expressed in the E. coli Origami/pET 32b system.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

et al. 2004

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Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 g per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.

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“…Often, the main purpose of expressing a recombinant protein in bacteria is to accumulate soluble product in the cytoplasm (Villaverde and Carrio 2003). In the Origami strains from Novagen, the disulfide bond-dependent folding of heterologous proteins is improved via disruption of the trxB and gor genes fused to the N-terminal of pET 32b plasmid sequences that encode two reductase enzymes, which allows for the formation of disulfide bonds in the E. coli cytoplasm (Bessette et al 1999;Lehmann et al 2003;Stewart et al 1998).…”

Section: Introductionmentioning

confidence: 99%

“…This select method allows the high-throughput purification of hundreds of scFvs with yields in the range of 50-100 lg; the purity is sufficient evaluation in a cell-based proliferation assay. The properly folded recombinant protein has been characterized by flow cytometry and immunofluorescence in several studies (Lehmann et al 2003;Singh et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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

Mahgoub

2012

Biochem Genet

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Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.

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High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Cited by 54 publications

References 30 publications

The Basophil-Specific Ectoenzyme E-NPP3 (CD203c) as a Marker for Cell Activation and Allergy Diagnosis

The Basophil-Specific Ectoenzyme E-NPP3 (CD203c) as a Marker for Cell Activation and Allergy Diagnosis

Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

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

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