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Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification

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Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2702))

Abstract

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-β1).

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References

  1. Smith GP (2019) Phage display: simple evolution in a petri dish (nobel lecture). Angew Chem Int Ed Engl 58:14428–14437. https://doi.org/10.1002/anie.201908308

    Article  CAS  PubMed  Google Scholar 

  2. Ling MM (2003) Large antibody display libraries for isolation of high-affinity antibodies. Comb Chem High Throughput Screen 6:421–432

    Article  CAS  PubMed  Google Scholar 

  3. Perelson AS, Oster GF (1979) Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination. J Theor Biol 81:645–670. https://doi.org/10.1016/0022-5193(79)90275-3

    Article  CAS  PubMed  Google Scholar 

  4. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314

    Article  CAS  PubMed  Google Scholar 

  5. Gründemann D, Schömig E (1996) Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898–903. https://doi.org/10.2144/96215rr02

    Article  PubMed  Google Scholar 

  6. Scholle MD, Kehoe JW, Kay BK (2005) Efficient construction of a large collection of phage-displayed combinatorial peptide libraries. Comb Chem High Throughput Screen 8:545–551

    Article  CAS  PubMed  Google Scholar 

  7. Weiss GA, Watanabe CK, Zhong A, Goddard A, Sidhu SS (2000) Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc Natl Acad Sci U S A 97:8950–8954. https://doi.org/10.1073/pnas.160252097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382. https://doi.org/10.1016/0076-6879(87)54085-x

    Article  CAS  PubMed  Google Scholar 

  9. Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7:e52031. https://doi.org/10.1371/journal.pone.0052031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Holland EG, Acca FE, Belanger KM, Bylo ME, Kay BK, Weiner MP, Kiss MM (2015) In vivo elimination of parental clones in general and site-directed mutagenesis. J Immunol Methods 417:67–75. https://doi.org/10.1016/j.jim.2014.12.008

    Article  CAS  PubMed  Google Scholar 

  11. Huovinen T, Brockmann EC, Akter S, Perez-Gamarra S, Yla-Pelto J, Liu Y, Lamminmaki U (2012) Primer extension mutagenesis powered by selective rolling circle amplification. PLoS One 7:e31817. https://doi.org/10.1371/journal.pone.0031817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Koide A, Bailey CW, Huang X, Koide S (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284:1141–1151. https://doi.org/10.1006/jmbi.1998.2238

    Article  CAS  PubMed  Google Scholar 

  13. Miller CJ, McGinnis JE, Martinez MJ, Wang G, Zhou J, Simmons E, Amet T, Abdeen SJ, Van Huysse JW, Bowsher RR, Kay BK (2021) FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein. New Biotechnol 62:79–85. https://doi.org/10.1016/j.nbt.2021.01.010

    Article  CAS  Google Scholar 

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Acknowledgments

The work was supported in part at the University of Illinois at Chicago, Loyola University, and Tango Biosciences by grant awards, U54DK093444, R21AI100565 and RO1AI100129, and 1R43AI157021, from the National Institutes of Health, respectively. The authors thank Dr. Leslyn Hanakahi (College of Pharmacy, University of Illinois, Chicago, Rockford) for the gift of Ku70/80 protein. We thank Drs. Emily Delaney and Nigel Delaney for assistance in analyzing the next-generation sequencing data for the library. We also thank Ms. Ashley Grahn and Ms. Grace Allen for editorial assistance in preparing this manuscript.

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Authors and Affiliations

  1. MacroGenics, Rockville, MD, USA

    Renhua R. Huang

  2. AstraZeneca, Gaithersburg, MD, USA

    Michael Kierny

  3. Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA

    Veronica Volgina & Makio Iwashima

  4. Tango Biosciences, Chicago, IL, USA

    Christina Miller & Brian K. Kay

Authors
  1. Renhua R. Huang
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  2. Michael Kierny
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  3. Veronica Volgina
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  4. Makio Iwashima
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  5. Christina Miller
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  6. Brian K. Kay
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Corresponding authors

Correspondence to Renhua R. Huang or Brian K. Kay .

Editor information

Editors and Affiliations

  1. Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Huang, R.R., Kierny, M., Volgina, V., Iwashima, M., Miller, C., Kay, B.K. (2023). Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification. In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_10

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  • DOI: https://doi.org/10.1007/978-1-0716-3381-6_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3380-9

  • Online ISBN: 978-1-0716-3381-6

  • eBook Packages: Springer Protocols

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