Protein Engineering Protocols - Mycobacteriology research center

Compartmentalized Self-Replication 239to select for pol η function in a RAD30, RAD52 yeast strain, which yielded amutant of pol η with increased activity (17).Phage display technology has been a highly successful method for repertoireselection, in particular, for the directed evolution of molecular interactions,allowing, for example, the isolation of peptide hormone mimics or specific antibodiesdirectly from repertoires of human V-genes. Recently, phage display hasbeen adapted for the selection of catalytic activity by proximal display of bothsubstrate and enzyme on the phage particle. This concept has been used successfullyby Jestin and Winter to enrich for active over inactive polymerases,relying on the in cis incorporation of a tagged nucleotide into a template–primerduplex substrate tethered to the phage particle (18). More recently, Romesbergand coworkers (19) used a similar approach to select for a variant of the Stoffelfragment that incorporates rNTPs with efficiencies approaching those of thewild-type enzyme for dNTP substrates.Although the genetic complementation approach is severely limited in theproperties that can be selected for, the phage methods seem much more versatile.However, selection conditions have to be compatible with phage viability,and the intramolecular tethering of the substrate may favor the selection ofpolymerases with a low affinity for the template–primer duplex and/or a poor processivity.However, both methods should potentially be able to detect extremelyweak polymerase activities, requiring, potentially, only a single dNTP incorporationfor selection.1.3. Polymerase Engineering by Compartmentalized Self-ReplicationWe have pursued another new strategy for the evolution of enzymes and, inparticular, polymerases, called compartmentalized self-replication (CSR; ref.20). In CSR, individual polymerase variants are isolated in separate compartments.Provided with appropriate reagents, each polymerase replicates only itsown encoding gene, to the exclusion of those in other compartments (i.e., itself-replicates; Fig. 1). Consequently, only genes encoding active polymerasesare replicated, whereas inactive variants disappear from the gene pool. Amongdifferentially active variants, the more active variants will make proportionallymore copies of their own encoding gene (i.e., will produce more “offspring”).As a result, the copy number of a polymerase gene after replication reflects thecatalytic activity of the polymerase it encodes under the given selection conditions.Thus, polymerase genes encoding the most active polymerases that arebest adapted to the selection conditions are going to increase in number and willcome to dominate the gene population.Segregation of self-replication into discrete, physically separate compartmentsis critical to ensure linkage of phenotype and genotype during CSR (i.e., to ensurethat adaptive gains of each polymerase only benefit the replication of its own