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Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method

  • Wen-Ching Lin
    Wen-Ching Lin
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
  • Hao-Cheng Tang
    Hao-Cheng Tang
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
  • Han Ying Wang
    Han Ying Wang
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
  • Chia-Yi Kao
    Chia-Yi Kao
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
    Taiwan International Graduate Program in Molecular Medicine, National Yang Ming Chiao Tung University and Academia Sinica, Taipei 11529, Taiwan
    More by Chia-Yi Kao
  • You-Chiun Chang
    You-Chiun Chang
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
    Taiwan International Graduate Program in Chemical Biology and Molecular Biophysics, National Taiwan University and Academia Sinica, Taipei 11529, Taiwan
  • Athena Hsu Li
    Athena Hsu Li
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
    Taiwan International Graduate Program in Interdisciplinary Neuroscience, National Yang Ming Chiao Tung University and Academia Sinica, Taipei 11529, Taiwan
  • Shi-Bing Yang
    Shi-Bing Yang
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
  • , and 
  • Kurt Yun Mou*
    Kurt Yun Mou
    Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
    *Email: [email protected]. Phone: +1-886-2-26523009. Fax: +1-886-2-26510774.
    More by Kurt Yun Mou
Cite this: ACS Synth. Biol. 2022, 11, 4, 1658–1668
Publication Date (Web):March 24, 2022
https://doi.org/10.1021/acssynbio.2c00086
Copyright © 2022 American Chemical Society

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    Abstract

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    Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.2c00086.

    • ssDNA preparation for antibody CDR randomization using the reverse Kunkel method; long sequence alignments of Fab variants generated by the reverse Kunkel method; ssDNA preparation for nanobody CDR randomization using the reverse Kunkel method; mutation analysis using a high/imbalanced protocol; function of the probability of all mutations occurred in the CDR regions versus the CDRs/FWRs mutational ratio; enrichment ratio test for the phage display selection against EGFP; sequence of the αGFP-#20 clone; fitting residual plots of the αGFP BLI experiments; characterization of αGFP-#20 in immunofluorescence experiments; enrichment ratio test for the phage display selection against NPY; single clone screening of the αNPY Fab yeast library; sequence of the αNPY-#C12 clone; fitting residual plots of the αNPY BLI experiments; comparison of αNPY-parental and αNPY-#C12 in immunofluorescence experiments using cell lines; comparison of the reverse Kunkel method and the traditional method for fragment-directed random mutagenesis; and step-by-step protocol of the reverse Kunkel method (PDF)

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    Cited By

    This article is cited by 1 publications.

    1. Fengjiao Zhao, Feng Chen, Huahang Yu, Siyue Fan, Min Bai, Jing Xue, Yue Zhao, Xiaolei Zuo, Chunhai Fan, Yongxi Zhao. CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction. Fundamental Research 2022, 47 https://doi.org/10.1016/j.fmre.2022.06.017

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