کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843670 | 1069287 | 2005 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
In this work, we describe the expression, purification, and disulfide mapping of the named 'peanut seed cDNA 33' (PSC33) peanut allergen. A variant of PSC33 (with N63, E64, Q69 instead of D63, Q64, E69) has been identified in peanut by proteomic analysis of a highly IgE immunoreactive purification fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and amplified it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally amplified. Since expression of the natural PSC33 (DQE) gene was very low in Escherichia coli even with supplementation of rare codon tRNAs, a synthetic gene optimized for expression in E. coli of PSC33 (DQE) was introduced into a pET9-c vector. A high production of protein occurred in the inclusion bodies that was submitted to refolding using an additive-introduced stepwise dialysis protocol which consists in the gradual removal of the denaturing agent guanidine-HCl with controlled introduction of oxidized and reduced glutathione and l-arginine as a chemical chaperone. After reverse phase HPLC purification, 1Â mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100Â ml of bacterial culture. Trypsin and CNBr hydrolysis of the protein combined with MALDI-TOF mass spectrometry allowed us to assign disulfide bridges and show that the native and refolded proteins were identical. The four disulfides of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 44, Issue 2, December 2005, Pages 110-120
Journal: Protein Expression and Purification - Volume 44, Issue 2, December 2005, Pages 110-120
نویسندگان
Gilles Clement, Didier Boquet, Lucie Mondoulet, Patricia Lamourette, Hervé Bernard, Jean Michel Wal,