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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
12:31

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Published on: May 1, 2018

Oligovalent Fab display on M13 phage improved by directed evolution.

Tuomas Huovinen1, Hanna Sanmark, Jani Ylä-Pelto

  • 1Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland. tuohuo@utu.fi

Molecular Biotechnology
|December 17, 2009
PubMed
Summary

Directed evolution improved antibody fragment (Fab) display on M13 phage. A specific mutation in the PelB signal sequence significantly enhanced Fab display 19-fold and increased phage titers twofold.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Efficient display of antibody fragments on filamentous phage M13 is essential for biopanning.
  • Improving the display of poorly performing antibody fragments (Fab) fused to phage gene-3 minor coat protein (g3p) is a key challenge.

Purpose of the Study:

  • To enhance the oligovalent display of a Fab fragment on filamentous phage M13 using directed evolution.
  • To identify and characterize mutations responsible for improved Fab display efficiency.

Main Methods:

  • A directed evolution strategy was employed using a randomly mutated Fab gene library.
  • Enrichment of Fab-displaying clones was achieved using polyclonal anti-mouse IgG antibodies.
  • The contribution of individual mutations to improved display was analyzed.

Main Results:

  • Two point mutations were identified as significantly contributing to improved Fab display efficiency.
  • A start codon mutation (AUG to GUG) in the PelB signal sequence of the heavy chain had the most substantial effect.
  • The engineered clone, FabM(GUG), exhibited a 19-fold improvement in Fab display and a twofold increase in phage titers.

Conclusions:

  • The PelB signal sequence start codon mutation is critical for enhancing Fab display on M13 phage.
  • This engineered Fab display system offers a more efficient platform for biopanning and antibody discovery.