An NMR-guided approach for identifying co-factors in boosting the Pfu DNA polymerase

Abstract

ABSTRACTWith rapid developments of emerging technologies like synthetic biology, the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly. Thus, rational optimization of the performance of DNA polymerase is of great interest. Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique used for studying protein structure and dynamics. It provides the atomic resolution information of enzymes under its functional solution environment to reveal the active sites (hot spots) of the enzyme, which could be further used for optimizing the performance of enzymes. Here we applied NMR spectroscopy to determine hot spots of the Pfu polymerase. Employing these hot spots as probes, two new co-factors, the heat shock protein TkHSP20 fromThermococcus Kodakaraensisand the chemical chaperone L-arginine, are identified to interact with Pfu polymerase to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu polymerase. This NMR-guided approach requires no prior assignment information of target enzymes, simplifying the exploration of novel co-factors for Pfu polymerase. Moreover, our approach is not dependent on structural data or bioinformatics. Therefore, it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.GRAPHICAL ABSTRACT