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A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

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Abstract

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations (>2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.

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Acknowledgments

We are grateful to Dr. Alexander Schenk, Andreea Scacoic, Marcus Schallmey and Dr. Hemanshu Mundhada for the technical support and fruitful scientific discussions. We also thank the Biokatalyse2021 Cluster and the German Ministry of Education and Research (BMBF) for the financial support.

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Authors and Affiliations

  1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Tsvetan Kardashliev, Anna Joëlle Ruff, Jing Zhao & Ulrich Schwaneberg

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  1. Tsvetan Kardashliev
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  2. Anna Joëlle Ruff
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  3. Jing Zhao
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  4. Ulrich Schwaneberg
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Correspondence to Ulrich Schwaneberg.

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Kardashliev, T., Ruff, A.J., Zhao, J. et al. A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis. Mol Biotechnol 56, 274–283 (2014). https://doi.org/10.1007/s12033-013-9706-0

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