Lisa D. Vailes, MSa, Michael T. Kinter, PhDb, L

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High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris Lisa D. Vailes, MSa, Michael T. Kinter, PhDb, L.Karla Arruda, MD, PhDa, Martin D. Chapman, PhDb Journal of Allergy and Clinical Immunology Volume 101, Issue 2, Pages 274-280 (February 1998) DOI: 10.1016/S0091-6749(98)70393-8 Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 1 Schematic outline of protein expression in P. pastoris. Journal of Allergy and Clinical Immunology 1998 101, 274-280DOI: (10.1016/S0091-6749(98)70393-8) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 2 Time course for expression of rBla g 4. Supernatants from rBla g 4 P. pastoris cultures were harvested at different time points and analyzed by Coomassie-stained SDS-PAGE. Cultures expressing albumin were used as positive controls. Journal of Allergy and Clinical Immunology 1998 101, 274-280DOI: (10.1016/S0091-6749(98)70393-8) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 3 Purification of rBla g 4 by chromatography on phenyl Sepharose. Lane 1, MW markers; Lane 2, culture supernatant; Lane 3, 1.5 mol/L NaCl eluted pool; Lane 4, 1.0 mol/L NaCl eluted pool; and Lane 5, high-performance liquid-chromatography purified rBla g 4. Journal of Allergy and Clinical Immunology 1998 101, 274-280DOI: (10.1016/S0091-6749(98)70393-8) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 4 Effect of deglycosylation on Pichia-expressed rBla g 4. Lane 1, MW markers; lane 2, untreated rBla g 4; lanes 3 and 4, Endo F–treated rBla g 4 (1 U and 2 U of enzyme, respectively); and lane 5, E. coli–expressed rBla g 4. Journal of Allergy and Clinical Immunology 1998 101, 274-280DOI: (10.1016/S0091-6749(98)70393-8) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 5 Inhibition RIA comparing the effect of deglycosylation on IgE antibody binding. Glycosylated or deglycosylated rBla g 4 from P. pastoris was used to inhibit binding of 125I-labeled Pichia rBla g 4 to IgE antibody in serum from an individual allergic to cockroaches. Journal of Allergy and Clinical Immunology 1998 101, 274-280DOI: (10.1016/S0091-6749(98)70393-8) Copyright © 1998 Mosby, Inc. Terms and Conditions