Protein Expression and PuriWcation 44 (2005) 110–120 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.05.015 Expression in Escherichia coli and disulWde bridge mapping of PSC33, an allergenic 2S albumin from peanut Gilles Clement a,¤ , Didier Boquet b , Lucie Mondoulet a , Patricia Lamourette b , Hervé Bernard a , Jean Michel Wal a a Laboratoire INRA-CEA d’immunoallergie alimentaire, SPI Bât 136 CEA, Saclay 91191, Gif sur Yvette Cedex, France b Service de Pharmacologie et d’Immunologie, SPI Bât 136 CEA, Saclay 91191, Gif sur Yvette Cedex, France Received 10 March 2005, and in revised form 24 May 2005 Available online 24 June 2005 Abstract In this work, we describe the expression, puriWcation, and disulWde 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 identiWed in peanut by proteomic analysis of a highly IgE immunoreactive puriWcation fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and ampliWed it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally ampliWed. 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 puriWcation, 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 disulWde bridges and show that the native and refolded proteins were identical. The four disulWdes of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.  2005 Elsevier Inc. All rights reserved. Keywords: Peanut allergen; Ara h 6; 2S albumin; Escherichia coli; Refolding; Additive-introduced stepwise dialysis; DisulWde bridge mapping 2S albumins are seed storage proteins found in dicot- yledonous plants and particularly in legumes. They share with cereals -amylase/trypsin inhibitors and non-spe- ciWc lipid transfer proteins (nsLTP) 1 a characteristic four helix, four disulWde bridges structure which has proved of great utility in plant evolution by providing respec- tively food for the embryo, protection from predators, and lipid transfer [1,2]. This structure provides compact- ness and resistance to proteolysis and this might be why a high number of plant allergens have it. The prolamin superfamily to which these three subfamilies belong counts 39 members out of 133 plant allergens recently compilated and as such is the bigger family of plant allergens [3]. Nine peanut allergens have been cloned (Ara h 1–8 and oleosin) and the corresponding proteins have all been isolated except Ara h 7 [4]. Not only the 2S is the albumin family represented (Ara h 2, 6, 7), but also 7S globulins (Ara h 1), 11S globulins (Ara h 3, 4), proWlin * Corresponding author. Fax: +33 1 69 08 59 07. E-mail address: gilles.clement@cea.fr (G. Clement). 1 Abbreviations used: nsLTP, non-speciWc lipid transfer proteins; CHCA, -cyano-hydroxy-cinnamic acid; TFA, triXuoroacetic acid; IPTG, isopropylthio--D-galactoside; DTT, dithiothreitol; Gu–HCl, guanidine–HCl; CD, circular dichroism.