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© Institut Pasteur
Cristaux de cellulase, enzyme purifiée de Clostridium thermocellum permettant la digestion de la cellulose. Image colorisée.
Publication : Biocatalysis and Biotransformation

Iterative cycles of in vitro protein selection for DNA polymerase activity

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Biocatalysis and Biotransformation - 14 Apr 2003

Sophie Vichier-Guerre, jean-Luc Jestin

Biocatal. Biotransfor. 2003;21(2):75-78

solation of genes encoding catalysts for defined chemical reactions should be facilitated by selection of proteins for catalysis from molecular repertoires. Display of proteins on phage allows the coupling between a protein and its gene. Furthermore, if the reaction product can be linked to the phage enzyme catalyzing the reaction, affinity chromatography for the product yields the protein catalyzing the reaction and its gene. One main advantage of this selection method is that it can be in principle generalized to most chemical reactions.

Here, iterative in vitro selections for polymerase activity were used to isolate a single phage-polymerase among more than 10(8) phage particles. Three to five selection cycles were required depending on the fraction of infectious phage-polymerases in the initial phage population. This is the first report quantifying the enrichment over iterative selection rounds for the isolation of rare catalysts displayed on phage.

http://www.tandfonline.com/doi/abs/10.1080/102424203100011085