High salt resistance lambda exonuclease engineering: electrostatic modulation of DNA interacting loop

Abstract

Highly processive exonucleases have a wide range of applications in the field of biological research. It is important to maintain this processivity under various conditions. Lambda exonuclease is one of the enzymes with the strongest known exonuclease processivity. However, the reaction conditions for lambda exonuclease are limited by factors such as pH and salt concentration. Enabling lambda exonuclease to function in various extreme conditions would open a new chapter for the enzyme to be used in industry and research fields. In this study, we aim to stabilize the protein-DNA interaction through Arg45 related, positive enhanced mutations to prevent loss of activity over a wider range of salt concentrations. To achieve this, several residues located near the Arg45 residues were changed to positive charges with Arginine substitution. We made one or two additional arginine substitution near the Arg45 residue and glutamic acid or even deletion mutation of Arg45 to see the effect of the charge of the loop when it comes to the activity in high salt condition of the enzyme. As a result, when interaction between protein and DNA is reinforced by introducing positive charge enhanced mutations. Activity was found not only under normal reaction conditions, but also high salt concentration as 300mM KCl in the positive charge enhanced mutations, and not in wildtype or negative charged mutation. The affinity of the enzyme with DNA substrate of the positive charged mutation was also stronger than the wildtype. This suggests that DNA-protein interaction in conventional lambda exonuclease was hindered by high salt concentrations and the activity under extreme conditions could be maintained by bypassing salt concentration-dependent processes through protein engineering to reinforce charge interaction between Arg45 near residues and DNA. This strategy can be extended to maintain salt stability for several enzymes that operate with charge interaction.