Automating Gene Library Synthesis by Structure-Based Combinatorial Protein Engineering

摘要: Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations domains structure. SCOPE was originally inspired DNA shuffling, which mimics during meiosis, where parental genes are "shuffled" novel in the resulting progeny. shuffling utilizes sequence identity between mediate template-switching events (the annealing and extension one fragment on another) PCR reassembly reactions generate crossovers hence genes. In light conservation structure degeneracy sequence, developed enable "shuffling" distantly related with no requirement for identity. The central principle involves use oligonucleotides encode regions choreograph assembly fragments chimeric This approach initially hybrid polymerases parents, later mutant library sesquiterpene synthases explore catalytic landscapes underlying functional divergence enzymes. chapter presents simplified protocol that can be integrated different mutagenesis techniques suitable automation liquid-handling robots. Two examples presented illustrate application using plant as model system. first example, we outline how an active-site series complex mixtures diverse mutants. second focused array individual clones distil minimal functionally important mutations. Through these examples, principles technique illustrated suitability automating various aspects procedure given applications discussed.