title: Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli.
abstract: Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.
authors.affiliation: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, Kuala Lumpur, 56000, Malaysia. kelvinlew86213@yahoo.com.
authors.forename: Min Han
authors.initials: MH
authors.lastname: Lew
authors.affiliation: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, Kuala Lumpur, 56000, Malaysia. reneelim@ucsiuniversity.edu.my.
authors.forename: Renee Lay Hong
authors.initials: RL
authors.lastname: Lim
mesh_terms: D055516:2S Albumins, Plant / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000485:Allergens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002846:Chromatography, Affinity; D003062:Codon; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D051379:Mice; D021183:Peanut Hypersensitivity / Q000175:diagnosis / Q000276:immunology / Q000628:therapy; D010940:Plant Proteins; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; D003062:Codon; D000939:Epitopes; D006023:Glycoproteins; D007074:Immunoglobulin G; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E
keywords: Ara h 2.02; Codon optimise; Escherichia coli; Peanut allergen; pET-28a (+) vector