Random mutagenesis and phage display technology as a tool for identifying ige epitopes of the birch pollen allergen Bet v 1

摘要

BackgroundIgE-binding epitopes of the major birch pollen allergen Betv 1 are dependent on the native structure of the allergen.To date, only a single IgE epitope has been identified. Weaimed to identify additional epitopes by directed in vitroevolution of non-IgE-binding structural homologues toproteins carrying single IgE-binding patches. For that purpose,we chose cytokinin-specific binding protein (CSBP)from Vigna radiata (mung bean), a structural homologueof Bet v 1 with only 31% sequence identity, as a templatesubjected to a combination of random mutagenesis andphage display.MethodsCSBP was expressed in Escherichia coli and purified bychromatographic methods. The protein integrity was verifiedby SDS-PAGE, mass spectrometry and circulardichroism and IgE binding assessed by ELISA using serafrom a panel of birch pollen allergic patients. Randommutagenesis of CSBP was performed by PCR using mutagenicnucleotide analogues. Phage display libraries of randomlymutated CSBP were created in the filamentousphage M13 by inserting PCR products into the phagmidpTP127. Biopanning was performed for selecting phageswith binding activity to birch pollen allergic patients’ IgE.After each panning round, an additional mutagenesis wasperformed and a new library created. IgE binding activitiesof enriched phages and of bacterial colonies representingsingle clones were analysed by transfer to nitrocelluloseand immunostaining.ResultsPurified recombinant CSBP revealed a secondary structurewith high similarity to that of Bet v 1, but only low IgEbinding in 11 of 35 sera from Bet v 1-sensitised birch pollenallergic patients. Phage libraries CSBPm1, CSBPm2and CSBPm3 with diversities of 105-107 different cloneswere created. Sequencing of 19 randomly picked clonesfrom the unselected CSBPm1 library showed an averagemutation rate of 5% (range 2.5-11.5). Analysis of singleclones from the first two panning rounds yielded cloneswhich expressed IgE binding mutant proteins.ConclusionDirected in vitro evolution of CSBP by random mutagenesisof surface residues might be a suitable tool fordefining conformational IgE binding epitopes of thebirch pollen allergen Bet v 1.This study was supported by grant P-22559-B11 fromthe Austrian Science Fund.