Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin).
BACKGROUND
Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua.


OBJECTIVE
We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy.


METHODS
Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen.


RESULTS
Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin.


CONCLUSIONS
This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.
Vallverdú
A
A
IFIDESA-ARISTEGUI, Research and Development Department, Bilbao, Spain.
Asturias
J A
JA
Arilla
M C
MC
Gómez-Bayón
N
N
Martínez
A
A
Martínez
J
J
Palacios
R
R
D000485:Allergens / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001249:Asthma / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003285:Contractile Proteins; D003429:Cross Reactions / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007525:Isoelectric Focusing; D008840:Microfilament Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011058:Pollen / Q000276:immunology; D016133:Polymerase Chain Reaction; D051304:Profilins; D011994:Recombinant Proteins / Q000276:immunology; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D003285:Contractile Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D000939:Epitopes; D008840:Microfilament Proteins; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E