Characterisation of a DNA polymerase highly mutated along the template binding interface.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Phage display is a powerful technique for linking genes to their encoded proteins, widely used for isolating molecular targets.
• A novel approach links phage-displayed enzymes to their reaction products, enabling selection of catalytically active enzymes and their genes via affinity chromatography.

Purpose of the Study:

• To apply the phage display strategy for enzyme selection to evolve a novel DNA polymerase.
• To characterize the kinetic properties and template specificity of the evolved polymerase.

Main Methods:

• Directed evolution of Thermus aquaticus DNA polymerase I using a phage display system.
• Selection of polymerase variants based on their catalytic activity and product formation.
• Kinetic characterization of the selected enzyme variant.

Main Results:

• A Thermus aquaticus DNA polymerase I variant with 15 mutations was successfully isolated.
• The evolved polymerase exhibits broad template specificity.
• The enzyme functions as a thermostable DNA-dependent and RNA-dependent DNA polymerase.

Conclusions:

• The phage display strategy is effective for the directed evolution of enzymes with novel functionalities.
• The selected polymerase variant represents a significant advancement in enzyme engineering, offering dual DNA and RNA templating capabilities.
• This engineered enzyme has potential applications in various molecular biology techniques requiring thermostable polymerases.