Abstract
A peptide Phage Display library was constructed using the BTK2 toxin as scaffold and rolling cycle amplification combined with Kunkel mutagenesis for rational diversification. During phage-display library construction, the persistence of wild type sequence in the library is a problem since prevents the achievement of highly diverse libraries, and may cause selection problems due to clonal expansion advantages of the wild type sequence over specific clones. Therefore, the assurance of 100% mutagenic primer incorporation during Kunkel reactions is of great impact for improved library construction strategies. In order to study improvements on oligo incorporation during Kunkel mutagenesis, we tested different mutagenic primers differing on the length of flanking annealing regions as well as the extension of mutagenic loop, which improved markedly the respective incorporation rates. In our studies, target diversification varied from 14 to 100% primer incorporation depending on specific primer features. Elongated loop encompassing the mutagenic region allowed diminished steric hindrance and complete annealing, which resulted in complete primer incorporation. Such technical advances for the production of fully diverse phage display libraries are crucial for the construction of powerful libraries granting the development of new and potent ligands of biologically important targets.
Published Version
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