title: Auto-induction for high yield expression of recombinant novel isoallergen tropomyosin from King prawn (Melicertus latisulcatus) for improved diagnostics and immunotherapeutics.
abstract: Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-D-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens.
authors.affiliation: Molecular Immunology Group, James Cook University, Townsville, QLD, Australia; Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australia; Comparative Genomic Centre, James Cook University, Townsville, QLD, Australia; Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia.
authors.forename: Martina
authors.initials: M
authors.lastname: Koeberl
authors.affiliation: Molecular Immunology Group, James Cook University, Townsville, QLD, Australia; Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australia; Comparative Genomic Centre, James Cook University, Townsville, QLD, Australia; Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia.
authors.forename: Sandip D
authors.initials: SD
authors.lastname: Kamath
authors.affiliation: Molecular Immunology Group, James Cook University, Townsville, QLD, Australia; Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australia; Comparative Genomic Centre, James Cook University, Townsville, QLD, Australia; Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia.
authors.forename: Shruti R
authors.initials: SR
authors.lastname: Saptarshi
authors.affiliation: Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australia; Queensland Tropical Health Alliance, James Cook University, Cairns, QLD, Australia; Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smout
authors.affiliation: Department of Immunology, Monash University, Melbourne, Victoria, Australia; Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Victoria, Australia.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Rolland
authors.affiliation: Department of Immunology, Monash University, Melbourne, Victoria, Australia; Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Victoria, Australia.
authors.forename: Robyn E
authors.initials: RE
authors.lastname: O'Hehir
authors.affiliation: Molecular Immunology Group, James Cook University, Townsville, QLD, Australia; Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australia; Comparative Genomic Centre, James Cook University, Townsville, QLD, Australia; Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia. Electronic address: andreas.lopata@jcu.edu.au.
authors.forename: Andreas L
authors.initials: AL
authors.lastname: Lopata
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005512:Food Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D012758:Shellfish / Q000032:analysis*; D013045:Species Specificity; D014335:Tropomyosin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000485:Allergens; D011994:Recombinant Proteins; D014335:Tropomyosin
keywords: Allergy diagnostics; Auto-induction; Escherichia coli strains (K and B); King prawn; Recombinant allergen; Tropomyosin