Structure-based combinatorial protein engineering

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

Abstract: Publisher Summary This chapter discusses the gene library synthesis by structure-based combinatorial protein engineering (SCOPE). SCOPE is a process for the synthesis of gene libraries that lay the genetic foundation for the exploration of the relationship between structure and function in the encoded proteins. The comparative analysis of both structurally and functionally of protein primary, secondary, and tertiary structure generates numerous hypotheses to probe the relationship between molecular structure and the ensuing functional readout. SCOPE was applied to create a library representing all possible combinations of nine point mutations in the terpene cyclase, tobacco 5-epi-aristolochene synthase (TEAS). The location of mutations in the amino acid and nucleotide sequences of TEAS are presented. Over 600 colonies from discrete mixtures, representing about half of the complexity of the TEAS library, were picked and their sequences determined. It is found that aside from the low-level appearance of wild-type sequence and random mutations likely arising from PCR errors, the actual distribution of mutations in a given mixture was as designed experimentally.