Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut

• Published in: Protein expression and purification Vol. 44; no. 2; pp. 110 - 120
• Main Authors: Clement, Gilles, Boquet, Didier, Mondoulet, Lucie, Lamourette, Patricia, Bernard, Hervé, Wal, Jean Michel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2005
• Subjects: 2S albumin; 2S Albumins, Plant; Additive-introduced stepwise dialysis; Allergens - biosynthesis; Allergens - chemistry; Allergens - isolation & purification; Amino Acid Sequence; Antigens, Plant; Ara h 6; Arachis - chemistry; Arachis - genetics; Arachis hypogaea; Base Sequence; Binding, Competitive; Circular Dichroism; Cystine - analysis; Disulfide bridge mapping; Escherichia coli; Escherichia coli - genetics; Genes, Synthetic - genetics; Genetic Vectors - genetics; Immunoenzyme Techniques; Molecular Sequence Data; Peanut allergen; Plant Proteins - biosynthesis; Plant Proteins - immunology; Plant Proteins - isolation & purification; Polymerase Chain Reaction; Protein Folding; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Refolding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; 2S albumin; Escherichia coli; Refolding; Ara h 6; Peanut allergen; Additive-introduced stepwise dialysis; Disulfide bridge mapping
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.05.015

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 N 63, E 64, Q 69 instead of D 63, Q 64, E 69) 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, 1 mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100 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.