Protein Engineering Protocols - Mycobacteriology research center

14Compartmentalized Self-ReplicationA Novel Method for the Directed Evolutionof Polymerases and Other EnzymesFarid J. Ghadessy and Philipp HolligerSummaryCompartmentalized self-replication (CSR) is a novel method for the directed evolution ofenzymes and, in particular, polymerases. In its simplest form, CSR consists of a simple feedbackloop involving a polymerase that replicates only its own encoding gene (self-replication). Selfreplicationoccurs in discrete, spatially separate, noncommunicating compartments formed by aheat-stable water-in-oil emulsion. Compartmentalization ensures the linkage of phenotype andgenotype (i.e., it ensures that each polymerase replicates only its own encoding gene to the exclusionof those in the other compartments). As a result, adaptive gains by the polymerase directly(and proportionally) translate into genetic amplification of the encoding polymerase gene. CSRhas proven to be a useful strategy for the directed evolution of polymerases directly from diverserepertoires of polymerase genes. In this chapter, we describe some of the CSR protocols usedsuccessfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties,such as increased thermostability or resistance to the potent inhibitor, heparin, from arepertoire of randomly mutated Taq polymerase genes.Key Words: Compartmentalized self-replication; directed evolution; in vitro selection; emulsion;Taq polymerase.1. IntroductionThe ability to self-replicate, to faithfully copy the genome, is a defining characteristicof all life. In present day organisms, this fundamental process is carriedout by members of a diverse class of enzymes: the polynucleotide polymerases (1).Apart from genome replication, polynucleotide polymerases perform a range ofcore functions within the cell, including transcription, DNA repair, and telomeremaintenance. Recently, it was found that specialized polymerases are also involvedin a range of diverse processes, ranging from adaptive mutation and antibodyFrom: Methods in Molecular Biology, vol. 352: Protein Engineering ProtocolsEdited by: K. M. Arndt and K. M. Müller © Humana Press Inc., Totowa, NJ237