Display of functional nucleic acid polymerase onEscherichia colisurface and its application in directed polymerase evolution

摘要

We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2 '-O-methyl nucleotide triphosphates (2 '-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coliadhesin involved in diffuse adherence andPseudomonas aeruginosaesterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) ofE. coliDNA polymerase I on the surface ofE. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2 '-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2 '-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.