Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II.
The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.
Sidoli
A
A
Dipartimento di Ricerca Biologica e Tecnologica, Istituto Scientifico H San Raffaele, Milano, Italy.
Tamborini
E
E
Giuntini
I
I
Levi
S
S
Volonté
G
G
Paini
C
C
De Lalla
C
C
Siccardi
A G
AG
Baralle
F E
FE
Galliani
S
S
D000485:Allergens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D006636:Histamine Release; D006801:Humans; D008129:Lolium / Q000276:immunology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012882:Skin Tests
D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; C056754:Lol p II protein, Lolium perenne; D010940:Plant Proteins; D011994:Recombinant Proteins