title: Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli.
abstract: In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified - Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3' and 5' RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.
authors.affiliation: Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Wen-Che
authors.initials: WC
authors.lastname: Tsai
authors.affiliation: Department of Pediatrics, Taipei Veterans General Hospital, and School of Medicine, National Yang Ming University, Taipei, 112, Taiwan.
authors.forename: Tzee-Chung
authors.initials: TC
authors.lastname: Wu
authors.affiliation: Department of Pediatrics, National Taiwan University Hospital, National Taiwan University, Taipei, 100, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, 100, Taiwan; Department of Medical Research, National Taiwan University Hospital, Taipei, 100, Taiwan.
authors.forename: Bor-Luen
authors.initials: BL
authors.lastname: Chiang
authors.affiliation: Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan. Electronic address: hwwen@dragon.nchu.edu.tw.
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Wen
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D031022:Mangifera / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000485:Allergens; D010940:Plant Proteins; D011994:Recombinant Proteins
keywords: Allergen; Escherichia coli; Man i 1; Mango