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Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen
journal contributionposted on 1996-08-01, 00:00 authored by P M Smith, Cenk SuphiogluCenk Suphioglu, I J Griffith, K Theriault, R B Knox, M B Singh
Background: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type 1 allergy. Objective: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. Methods: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. Results: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. Conclusion: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.