Research

Molecular Biotechnology

, Volume 46, Issue 1, pp 58-62

Characterisation of a DNA Polymerase Highly Mutated Along the Template Binding Interface

  • Sophie Vichier-GuerreAffiliated withDépartement de Biologie Structurale et Chimie, Institut Pasteur
  • , Jean-Luc JestinAffiliated withDépartement de Biologie Structurale et Chimie, Institut Pasteur Email author 

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Abstract

Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant’s broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.

Keywords

Reverse transcriptase Enzyme engineering In vitro selection Phage display Directed evolution