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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution.

Aurelio Hidalgo1, Anna Schliessmann, Rafael Molina

  • 1Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Ernst-Moritz-Arndt University Greifswald, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany.

Protein Engineering, Design & Selection : PEDS
|June 19, 2008
PubMed
Summary

Focused directed evolution offers a new protein engineering approach. A novel PCR technique enables targeted gene fragment randomization, enhancing enzyme efficiency and substrate selectivity.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Traditional protein engineering methods include rational design and random mutagenesis (directed molecular evolution).
  • A gap exists for methods that combine targeted randomization with scaffold preservation.

Purpose of the Study:

  • To introduce a novel focused directed evolution technique for protein engineering.
  • To develop an efficient PCR method for randomizing specific gene fragments while maintaining the protein scaffold.

Main Methods:

  • Developed a PCR technique utilizing long, spiked oligonucleotides for targeted gene fragment randomization.
  • Applied the method to create focused mutant libraries of Pseudomonas fluorescens esterase I (PFEI).
  • Screened libraries for altered substrate selectivity and compared results with error-prone PCR libraries.

Main Results:

  • Achieved over 95% mutation incorporation efficiency, regardless of mutation position.
  • Generated large mutant libraries with the potential for simultaneous multi-locus randomization and recombination.
  • Identified two PFEI variants with a 10-fold increase in catalytic efficiency towards p-nitrophenyl dodecanoate.

Conclusions:

  • The developed PCR technique is highly efficient for focused directed evolution.
  • This method facilitates the discovery of enzyme variants with improved properties, such as enhanced catalytic efficiency and altered substrate selectivity.
  • Structural modeling of identified variants provides insights into the observed functional improvements.