Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen☆,☆☆,★,★★
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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Cited by (0)
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From aSchool of Botany, University of Melbourne, Parkville, Victoria; and bImmuLogic Pharmaceutical Corporation, Waltham.
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Supported by The National Health and Medical Research Council of Australia, Asthma Foundation of Victoria and Baker Shaw Trust.
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Reprint requests: Cenk Suphioglu, PhD, School of Botany, University of Melbourne, Parkville, Victoria, 3052, Australia.
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