Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells.
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 Ni2+ 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 decreases 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.
Rogers
B L
BL
ImmuLogic Pharmaceutical Corporation, Waltham, MA 02154.
Morgenstern
J P
JP
Garman
R D
RD
Bond
J F
JF
Kuo
M C
MC
D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells; D001483:Base Sequence; D002415:Cats / Q000276:immunology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology*; D013917:Thrombin / Q000378:metabolism; D014170:Transformation, Genetic
D000485:Allergens; D006023:Glycoproteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D013917:Thrombin; C059296:Fel d 1 protein, Felis domesticus