Recombinant fel d I: expression, purification, IgE binding and reaction with cat-allergic human T cells
| Autor: | Kuo Mei-Chang, Bruce L. Rogers, Julian F. Bond, Jay P. Morgenstern, Garman Richard D |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Hypersensitivity
Immediate Antigenicity T-Lymphocytes T cell Molecular Sequence Data Immunology Dose-Response Relationship Immunologic Antigen-Presenting Cells Enzyme-Linked Immunosorbent Assay Lymphocyte Activation Immunoglobulin E Cleavage (embryo) Polymerase Chain Reaction Chromatography Affinity law.invention Transformation Genetic Affinity chromatography law Escherichia coli medicine Animals Humans Amino Acid Sequence Molecular Biology Chromatography High Pressure Liquid Glycoproteins chemistry.chemical_classification Base Sequence biology Chemistry Thrombin Allergens Molecular biology Recombinant Proteins Amino acid medicine.anatomical_structure Biochemistry Cats biology.protein Recombinant DNA Oligonucleotide Probes Linker |
| Zdroj: | Molecular Immunology. 30:559-568 |
| ISSN: | 0161-5890 |
| DOI: | 10.1016/0161-5890(93)90030-f |
| Popis: | This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat ( Felis domesticus ). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni 2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR↓GS) at the N -terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N -terminus of the native sequence. Amino acid sequencing analysis of the N -terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen. |
| Databáze: | OpenAIRE |
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