Patient-tailored cloning of allergens by phage display: peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA.
A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10(11) cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-(L-proline)-Sepharose and yielded 30 mg l(-1) culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.
Kleber-Janke
T
T
Research Center, Borstel, Germany. t.kleber-janke@biophage.de
Crameri
R
R
Scheurer
S
S
Vieths
S
S
Becker
W M
WM
D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D010367:Arachis / Q000737:chemistry*; D001435:Bacteriophages / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003285:Contractile Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D051304:Profilins; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D003285:Contractile Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D051304:Profilins; D012333:RNA, Messenger; D011994:Recombinant Proteins