Angel Vallverdú, BSc, Juan A. Asturias, PhD, M

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Presentation transcript:

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin) Angel Vallverdú, BSc, Juan A. Asturias, PhD, M.Carmen Arilla, PhD, Nuria Gómez-Bayón, Alberto Martínez, PhD, Jorge Martínez, PhD, Ricardo Palacios, PhD Journal of Allergy and Clinical Immunology Volume 101, Issue 3, Pages 363-370 (March 1998) DOI: 10.1016/S0091-6749(98)70249-0 Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG.1. Complete nucleotide and amino acid–deduced (in bold) sequences of Mer a 1 (clone M2). Differences are shown only in clone M1 sequence, with corresponding amino acid change when it occurs. Primers used for cDNA amplification are underlined. The vertical arrow indicates the length of the C-truncated profilin. The nucleotide sequence of clone M2 has been submitted to the GeneBank/European Molecular Biology Laboratory (EMBL) Databases under accession number Y13271. Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG.2. Comparison of Mer a 1 with different plant profilins: Olea europaea profilin 1 (Y12425), Betula verrucosa (M65179), Nicotiana tabacum (X82120), Arabidopsis thaliana profilin 4 (U43324), Triticum aestivum profilin 1 (X89825), Hordeum vulgare (U49505), Phleum pratense (X77583), Zea mays profilin 2 (X73280), Cynodon dactylon (Y08390), and Phaseolus vulgaris (X81982). Points indicate identity of residues with respect to upper sequence. Structural and allergenic data obtained from birch and Arabidopsis profilins 15,36 are shown for comparison; secondary structure is represented in upper part of sequence. Important residues involved in conformational stability are in bold, and areas covering experimentally determined sequential IgE-reactive epitopes are underlined. Residues marked with asterisks are involved in the plant-specific binding pocket.36 Two points (:) indicate similar amino acids (A, S, T; D, E; N, Q; R, K; I, L, M, V; and F, Y, W). Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 3. Isoelectric focusing of M. annua–purified profilin. Lane 1, M FIG.3. Isoelectric focusing of M. annua–purified profilin. Lane 1, M. annua profilin; lane 2, pI markers. Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 4. Purification of recombinant Mer a 1 expressed in E. coli FIG.4. Purification of recombinant Mer a 1 expressed in E. coli. A, Twenty percent SDS-PAGE analysis of samples from the purification steps. B, Western blotting of A with rabbit anti-profilin serum. Lane M, molecular mass markers; lanes 1 and 2, total cell lysate from E. coli BL21 (DE3) containing pKN172 alone and pKN172 with Mer a 1 insert (clone M2), respectively; lanes 3 and 4, soluble and insoluble fractions of sample on lane 2, respectively; lane 5, purified recombinant Mer a 1 (2 μg); lanes 6 and 7, natural M. annua profilin (10 and 2 μg, respectively). C, mAb-reactivity to M. annua (E) and purified natural (P) and recombinant (R) Mer a 1. Lane M, molecular mass markers; lane 1, 7B12 mAb; lane 2, 6D2 mAb; lane 3, 5F2 mAb; lane 4, 10A4 mAb; lane 5, 11F11 mAb; lane 6, 3G4 mAb; lane 7, 1C12 mAb; and lane 8, 8F1 mAb. Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG.5.A, Competitive inhibition of binding of sunflower profilin rabbit IgG antibodies to solid-phase natural M. annua profilin by natural (open squares) or recombinant (filled squares) profilins from M. annua and the 15.3 kd profilin isoform isolated by electroelution (*). Control experiments were performed with BSA (filled inverted triangles). B, Competitive inhibition of binding of sunflower profilin rabbit IgG antibodies to solid-phase natural M. annua profilin by natural (open squares, circles, and triangles) or recombinant (filled circles and triangles) profilins from M. annua (open squares), Olea europaea (filled and open circles), and Cynodon dactylon (filled and open triangles). Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 6. Immunologic characterization of Mer a 1 FIG.6. Immunologic characterization of Mer a 1. IgE reactivity of sera from patients allergic to M. annua with M. annua extract (A) and purified recombinant Mer a 1 (B). Proteins transferred onto PVDF membranes were probed with sera from patients allergic to M. annua (lanes 1 through 9), serum pool from healthy individuals (lane 10), rabbit anti-profilin serum (lane 11), and buffer alone (lane 12). Molecular mass markers (lane M) were stained with Amido Black B. C, Mer a 1 immunoblot-inhibition. Protein extract from M. annua (M), O. europaea (O), and R. communis pollen (R) or purified latex profilin (L) were separated by SDS-PAGE and transferred onto PVDF membranes. Inhibition was performed by preincubating serum pools (L, O, and R) or serum (M) from patients sensitive to the corresponding extract with: lane 1, PBS buffer; lane 2, BSA; lane 3, natural profilin from latex (L), O. europaea pollen (O), or R. comunis pollen (R); lane 4, natural profilin from M. annua pollen; and lane 5, recombinant Mer a 1. Journal of Allergy and Clinical Immunology 1998 101, 363-370DOI: (10.1016/S0091-6749(98)70249-0) Copyright © 1998 Mosby, Inc. Terms and Conditions