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


CONCLUSIONS
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.
Smith
P M
PM
School of Botany, University of Melbourne, Parkville, Victoria, Australia.
Suphioglu
C
C
Griffith
I J
IJ
Theriault
K
K
Knox
R B
RB
Singh
M B
MB
D000485:Allergens; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000276:immunology*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006109:Poaceae / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C071778:CYND1 protein, Cynodon dactylon; D018076:DNA, Complementary; D015335:Molecular Probes; D010940:Plant Proteins