Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen.
BACKGROUND
Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of the crossreactivity found among pollen and food allergic patients. This allergen is usually under-represented in natural extracts used for allergy diagnosis.


OBJECTIVE
To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy.


METHODS
Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose.


RESULTS
Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced. Full-length C. dactylon profilin gene was expressed in Escherichia coli as non fusion protein. Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained. The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen. Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower profilin.


CONCLUSIONS
Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.
Asturias
J A
JA
IFIDSA-ARISTEGUI, Research and Development Department, Bilbao, Spain.
Arilla
M C
MC
Gomez-Bayon
N
N
Martinez
J
J
Martinez
A
A
Palacios
R
R
D000485:Allergens / Q000096:biosynthesis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006109:Poaceae; D011058:Pollen; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D010940:Plant Proteins