| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 102 === Despite growing evidence indicates the importance of loop in protein folding process, the hyper variety of loop sequence composition is still the major challenge for the traditional studies which are based on limited structural information to determine the functional key residues within the loop region. This conundrum can be resolved by a new strategy based on the combination of comprehensive mutational analysis, next generation sequencing (NGS), and high throughput functional assay. The tremendous data resulting from this strategy will be able to help us exploring the sequence-structure-function relationships in local protein regions effectively. The 6 CDRs and 10 non-CDR loops in variable domains of a model single chain variable fragment (scFv) which have been optimized with consensus sequence approach are used as our study platform. Different from previous studies based on numerous sets of single site saturated mutagenesis, we have constructed the scFv synthetic libraries with 6-7 amino acids mutated within the target loop region simultaneously. The results indicate that the essential sequences within the loop regions are dictated by the residues involving tertiary interactions, rather than the consensus sequences or the sequences with high turn propensities. Groups of variants with different sequence features from the wild type sequence are characterized by computational or experimental methods, and they reveal significant improvement in thermal stability and expression level. This study elaborates a new strategy for exploring the protein fitness landscape and possible sequence-function relationship unseen in nature.
|
|---|
