Enhanced immunoreactivity and preferential heterodimer formation of reassociated Fel d I recombinant chains.
In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing conditions, then allowed to reassociate by dilution and extensive dialysis against a physiological buffer. An initial examination of the reaction products using quantitative capture ELISA demonstrated comparable reactivity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel d I) also possessed an enhanced IgE binding capacity. Depletion ELISA results gave only a 5% difference in reactivity between rFel d I and the native protein versus a 20% difference with the mixture of the two chains. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. Here the results demonstrated no loss of reactivity with the reassociated chains as compared to Fel d I or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated fractions by mass spectrometry. It was clear from these results that like the native Fel d I, the products of the reassociation reaction favored heterodimer formation, with no homodimer being detected. This implies that the reassociated protein chains had preferentially adopted a native-like conformation.
Keating
K M
KM
ImmuLogic Pharmaceutical Corporation, Waltham, MA 02154, USA.
Segal
D B
DB
Craig
S J
SJ
Nault
A K
AK
Semensi
V
V
Wasserman
A S
AS
Counsell
C M
CM
Bond
J F
JF
D000818:Animals; D002415:Cats / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000378:metabolism; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D017510:Protein Folding; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus