title: High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris.
abstract: Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach-sensitive inner-city children. Bla g 4 is a ligand-binding protein (or calycin) that causes IgE antibody responses in 40% to 60% of patients allergic to cockroaches. Recombinant Bla g 4 was expressed in Escherichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/L culture). To improve yields, Bla g 4 was expressed in the Pichia pastoris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L. By cross-inhibition radioimmunoassay, Bla g 4 expressed in E. coli or P. pastoris provided overlapping inhibition curves. Both allergen preparations bound comparable levels of serum IgE antibody and showed similar skin test reactivity in individuals allergic to cockroaches (10[-1] to 10[-3] microg/ml). Deglycosylation of Pichia-expressed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet, and liquid chromatography-mass spectrometry results suggested that the 20 kd band contained residual sugar residues. Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE antibody binding in radioimmunoassay. Pichia-produced Bla g 4 had the same antigenic reactivity as that produced in E. coli, and glycosylation had no effect on IgE antibody binding. The high yield of Bla g 4 obtained in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma patients to cockroach allergens.
authors.affiliation: Department of Medicine, Asthma and Allergic Diseases Center, University of Virginia, Charlottesville 22908, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Vailes
authors.forename: M T
authors.initials: MT
authors.lastname: Kinter
authors.forename: L K
authors.initials: LK
authors.lastname: Arruda
authors.forename: M D
authors.initials: MD
authors.lastname: Chapman
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003058:Cockroaches / Q000276:immunology*; D006801:Humans; D019476:Insect Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012882:Skin Tests; D014170:Transformation, Genetic
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C097505:Blatella germanica allergen 4; D002352:Carrier Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins