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.

使用BTK2毒素作为支架构建了一个肽噬菌体展示文库，并将滚动周期扩增与Kunkel诱变相结合以实现合理的多样化。在噬菌体展示文库构建期间，由于阻碍了高度多样化文库的实现，文库中野生型序列的持久性是一个问题，并且由于野生型序列相对于特定克隆的克隆扩展优势，可能导致选择问题。因此，确保在Kunkel反应过程中100％诱变引物掺入对于改进文库构建策略具有重大影响。为了研究在Kunkel诱变过程中寡核苷酸掺入的改进，我们测试了不同的诱变引物，这些引物的侧翼退火区长度以及诱变环的延伸不同，这显着提高了各自的掺入率。在我们的研究中，靶标多样化取决于引物的具体特征，引物掺入量从14％到100％不等。包含诱变区的延长环可减少空间位阻，并完全退火，从而导致引物完全掺入。生产完全多样化的噬菌体展示文库的技术进步对于构建功能强大的文库至关重要，这些文库可开发出具有生物学重要目标的新型有效配体。这导致引物完全掺入。生产完全多样化的噬菌体展示文库的技术进步对于构建功能强大的文库至关重要，这些文库可开发出具有生物学重要目标的新型有效配体。这导致引物完全掺入。生产完全多样化的噬菌体展示文库的技术进步对于构建功能强大的文库至关重要，这些文库可开发出具有生物学重要目标的新型有效配体。