Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli

Ling, Chunfang; Zhang, Junyan; Chen, Huifang; Zou, Zehong; Lai, Hung-En; Zhang, Jianguo; Lin, Dan; Tao, Ailin

doi:10.1016/j.pep.2014.11.012

Cited by 5 publications

(4 citation statements)

References 35 publications

(44 reference statements)

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“…This finding indicates that this protein is expressed as an inclusion body and forms inert solid particles that are water‐insoluble. The E. coli expression system is the most commonly used and is convenient, but recombinant proteins often form inclusion bodies; size‐exclusion chromatography can help remove denatured substances in proteins, promote protein refolding, and separate folding intermediates 17 . We used nickel affinity column chromatography to obtain purified rDer p 22 to ensure robustness of our further in vitro and in vivo studies.…”

Section: Discussionmentioning

confidence: 99%

“…The E. coli expression system is the most commonly used and is convenient, but recombinant proteins often form inclusion bodies; size-exclusion chromatography can help remove denatured substances in proteins, promote protein refolding, and separate folding intermediates. 17 We used nickel affinity column chromatography to obtain purified innate lymphoid cells. 19,20 Increased Th2 cell levels correlate with an imbalanced Th1/Th2 ratio and IgE response, promoting the development of asthma.…”

Section: Rder P 22 Induces Apoptosis In Vitromentioning

confidence: 99%

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Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE‐binding in asthmatic children, and immunogenicity

Zhou

et al. 2022

Pediatric Allergy Immunology

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Background: Dust mite extract contains multiple components that, while useful in clinical allergy diagnosis and treatment, can cause serious side effects. Defining components of dust mite extract is important their contributions to allergic disease. This study aimed to characterize a novel dust mite allergen, Der p 22. Methods:We amplified the cDNA encoding Der p 22 from total RNA of the mite Dermatophagoides pteronyssinus, and inserted it into an expression construct for transformation to competent cells. Purified recombinant (r) Der p 22 was tested for IgEbinding reactivity in sera obtained from children with allergic asthma by the Affiliated Wuxi Children's Hospital of Nanjing Medical University (Jiangsu, China). rDer p 22 also was used to challenge BALB/c mice to assess effects on T helper cells and cytokine levels and applied to cultured lung epithelial cells to evaluate apoptosis and cytokine secretion.Results: rDer p 22 bound to IgE in 93.75% of sera from pediatric allergic asthma patients. Mice challenged with rDer p 22 had altered Th1/Th2 ratios in spleen and lymph, and lower levels of cytokines IFNγ but higher levels of IL-4 and IL-10 in alveolar lavage fluid compared with controls (p < .05). Cultured lung epithelial cells had greater apoptosis rates and exhibited higher levels of IL-6, IL-8, and GM-CSF when treated with rDer p 22 compared with control treatment (p < .05).Conclusions: Recombinant Der p 22 exhibited high IgE-binding rates in allergic children, indicating the activity of the recombinant protein and suggesting this novel allergen may be appropriate for inclusion in an allergy diagnostic workup. This finding is supported by in vitro and mouse in vivo studies showing rDer p 22 induced strong allergenic reactivity and apoptosis.

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Section: Discussionmentioning

confidence: 99%

Section: Rder P 22 Induces Apoptosis In Vitromentioning

confidence: 99%

Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE‐binding in asthmatic children, and immunogenicity

Zhou

et al. 2022

Pediatric Allergy Immunology

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“…The cleaved protein was isolated using a Superdex75 26/600 (Cytivia, Marlborough, MA) sizing column. The resulting protein was then incubated with 1 and 0.5 mM oxidized and reduced glutathione, respectively, to ensure the correct disulfide bonding pattern, , and exchanged into PBS using the Superdex75 26/600 sizing column to yield the final purified product. Protein concentrations were quantified using a BCA assay kit (Pierce Scientific, Rockford, IL).…”

Section: Methodsmentioning

confidence: 99%

Structure and IgE Cross-Reactivity among Cashew, Pistachio, Walnut, and Peanut Vicilin-Buried Peptides

Foo

Nesbit

Gipson

et al. 2023

J. Agric. Food Chem.

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Peanut and tree-nut allergies are frequently comorbid for reasons not completely understood. Vicilin-buried peptides (VBPs) are an emerging family of food allergens whose conserved structural fold could mediate peanut/tree-nut co-allergy. Peptide microarrays were used to identify immunoglobulin E (IgE) epitopes from the N-terminus of the vicilin allergens Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from three patient diagnosis groups: monoallergic to either peanuts or cashew/pistachio, or dual allergic. IgE binding peptides were highly prevalent in the VBP domains AH1.1, AO1.1, JR2.1, and PV3.1, but not in AO1.2, JR2.2, JR2.3, and PV3.2 nor the unstructured regions. The IgE profiles did not correlate with diagnosis group. The structure of the VBPs from cashew and pistachio was solved using solution-NMR. Comparisons of structural features suggest that the VBP scaffold from peanuts and tree-nuts can support cross-reactivity. This may help understand comorbidity and cross-reactivity despite a distant evolutionary origin.

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“…A1LS was purified from the resulting cells using an immobilized glutathione column, eluted with 10 mM reduced glutathione in pH 7.4 PBS, and incubated with TEV protease overnight to cleave the GST tag. Correct disulfide bond pairing was achieved by diluting the sample and adding oxidized glutathione to a final concentration of 2 mM, and 0.5 mM reduced and oxidized glutathione respectively [38] (redox buffer), the efficacy of which was subsequently verified using 1 H-15 N NMR and mass spec (S2), and further confirmed over the course of 3D structure determination described subsequently. Following 30 minute incubation the sample was loaded onto a Superdex75 26/600, and eluted with PBS to remove the GST tag.…”

Section: Constructs and Purificationmentioning

confidence: 98%

Structure and IgE cross-reactivity among walnut and peanut vicilin leader sequences

Nesbit

Gipson

et al. 2021

Preprint

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Background: Vicilin seed storage proteins are translated with N-terminal leader sequences (LSs) that are cleaved to yield the mature protein. These LSs were thought to be unstructured and rapidly degraded. However, Ara h 1 and Jug r 2 LS (A1LS, J2LS) have been identified in seeds, and immunodominant IgE epitopes detected. Here, common sequences containing structured CxxxC-repeat motifs were identified as potential mediators of IgE cross-reactivity despite very low (17%) sequence identity. Method: Linear IgE epitopes were identified by peptide microarrays, in which overlapping 15-mer peptides on glass slides, were incubated with sera from peanut, walnut or dual allergic individuals. Similar epitopes were computationally predicted. Peanut A1LS and walnut J2LS fragments (J2.1, J2.2, J2.3) each with a CxxxC vicilin LS motif were identified, cloned, expressed, purified and their structures solved using solution-NMR to locate and assess epitopes on the structure. Results: A1LS and J2LSs reveal similar helix-turn-helix motifs connected by disulfide bonds between adjacent CxxxC repeats forming α-hairpin structures. Peanut-allergic IgE bound more frequently to the J2LSs, regardless of walnut allergic status or A1LS binding. IgE binding pattern to peptides from both J2LS and A1LS, along with structure and computational predictions, suggest that the structure and conserved amino acid properties of peptides determine cross-reactivity. The properties of LS IgE epitopes were closely related to epitopes in 2S albumins. Conclusion: The shared α-hairpin structure is a stable scaffold that contributes to cross-reactivity despite low sequence identity. Biophysical properties are a better predictor of distant cross-reactivity than traditional measures of evolutionary conservation.

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Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli

Cited by 5 publications

References 35 publications

Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE‐binding in asthmatic children, and immunogenicity

Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE‐binding in asthmatic children, and immunogenicity

Structure and IgE Cross-Reactivity among Cashew, Pistachio, Walnut, and Peanut Vicilin-Buried Peptides

Structure and IgE cross-reactivity among walnut and peanut vicilin leader sequences

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