Purification and molecular cloning of a major allergen from Anisakis simplex.
A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.
Shimakura
Kuniyoshi
K
Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan. kunisan@s.kaiyodai.ac.jp
Miura
Hironori
H
Ikeda
Kaori
K
Ishizaki
Shoichiro
S
Nagashima
Yuji
Y
Shirai
Toshihiro
T
Kasuya
Shiro
S
Shiomi
Kazuo
K
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D017163:Anisakis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D017173:Caenorhabditis elegans / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics; D002845:Chromatography; D003001:Cloning, Molecular; D018509:DNA, Helminth / Q000737:chemistry / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000235:genetics
D000485:Allergens; D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D002135:Calcium-Binding Proteins; D018509:DNA, Helminth; D015801:Helminth Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C412511:troponin-like protein, Anisakis simplex; D007073:Immunoglobulin E; D014357:Trypsin