Chapter Two – Automating Gene Library Synthesis by Structure-Based Combinatorial Protein Engineering: Examples from Plant Sesquiterpene Synthases

Dokarry, Melissa, Laurendon, Caroline and O'Maille, Paul E. (2012) Chapter Two – Automating Gene Library Synthesis by Structure-Based Combinatorial Protein Engineering: Examples from Plant Sesquiterpene Synthases. Methods in Enzymology, 515. pp. 21-42. ISSN 0076-6879

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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.

Item Type:	Article
Uncontrolled Keywords:	terpene synthase,sesquiterpene,protein engineering,recombination,automation
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Pure Connector
Date Deposited:	24 Sep 2016 00:06
Last Modified:	22 Oct 2022 01:20
URI:	https://ueaeprints.uea.ac.uk/id/eprint/59813
DOI:	10.1016/B978-0-12-394290-6.00002-1

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