Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen,☆☆,,★★

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Abstract

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I 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. (J ALLERGY CLIN IMMUNOL 1996;98:331-43.)

Section snippets

Plant material

Bermuda grass pollen was obtained from Greer Laboratories Inc. (Lenoir, N.C.) as a dry, nondefatted pollen and stored at -20° C until required.

Monoclonal antibodies

Monoclonal antibodies 1D1, 3A2, and 4D2 were produced as described by Smith et al.12

Construction of cDNA libraries

RNA was isolated from Bermuda grass pollen by using a modification of the guanidinium thiocyanate method.17 After the pollen was ground in guanidinium thiocyanate buffer (5 mol/L guanidinium thiocyanate in 25 mmol/L sodium citrate [pH 7.0], 0.1 mol/L β-mercaptoethanol,

Isolation of cDNA clones encoding Cyn d 1

Monoclonal antibodies directed against Cyn d 1 were used to isolate cDNA clones encoding Cyn d 1. The preparation and specificity of these mAbs has been described elsewhere.12 Thirty-five positive clones were isolated from a Bermuda grass pollen cDNA expression library with mAb 3A2. These were retested to confirm binding to mAb 3A2, and eventually 28 positive clones were plaque-purified (Fig. 1). Proteins expressed by these cDNA clones were also tested for binding to mAb 4D2 (Fig. 1). Fusion

DISCUSSION

Through cDNA cloning and PCR amplification we have isolated clones encoding the major Bermuda grass pollen allergen Cyn d 1. The ORF of the clone encoding the complete Cyn d 1 polypeptide (CD1) encodes a protein of 246 amino acids with one possible N-glycosylation site. The predicted mass of the mature protein is 26.8 kd. This differs from that observed for major isoforms of the natural protein (31 and 32 kd), but the difference could be accounted for by glycosylation. Binding of concanavalin A

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    From aSchool of Botany, University of Melbourne, Parkville, Victoria; and bImmuLogic Pharmaceutical Corporation, Waltham.

    ☆☆

    Supported by The National Health and Medical Research Council of Australia, Asthma Foundation of Victoria and Baker Shaw Trust.

    Reprint requests: Cenk Suphioglu, PhD, School of Botany, University of Melbourne, Parkville, Victoria, 3052, Australia.

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