Skip to main content
SpringerLink
Log in

Purification and In Vitro Characterization of Zinc Finger Recombinases

  • Protocol
  • First Online:
Site-Specific Recombinases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1642))

Abstract

Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance. Here, we present a straightforward set of protocols for the expression and purification of ZFRs, an assay system for catalytic proficiency in vitro and bandshift assays for detection of sequence-specific DNA interactions.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Craig NL (2015) A moveable feast: an introduction to mobile DNA. In: Craig NL, Chandler M, Gellert M, Lambowitz A, Rice PA, Sandmeyer SB (eds) Mobile DNA III. ASM Press, Washington, DC

    Google Scholar 

  2. Olorunniji FJ, Rosser SJ, Stark WM (2016) Site-specific recombinases: molecular machines for the genetic revolution. Biochem J 473:673–684

    Article  CAS  PubMed  Google Scholar 

  3. Akopian A, Stark WM (2005) Site-specific recombinases as instruments for genomic surgery. Adv Genet 55:1–23

    CAS  PubMed  Google Scholar 

  4. Akopian A, He J, Boocock MR, Stark WM (2003) Chimeric recombinases with designed DNA sequence recognition. Proc Natl Acad Sci U S A 100:8688–8691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gordley RM, Gersbach CA, Barbas CF III (2009) Synthesis of programmable integrases. Proc Natl Acad Sci U S A 106:5053–5058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Carroll D (2014) Genome engineering with targetable nucleases. Annu Rev Biochem 83:409–439

    Article  CAS  PubMed  Google Scholar 

  7. Proudfoot CM, McPherson AL, Kolb AF, Stark WM (2011) Zinc finger recombinases with adaptable DNA sequence specificity. PLoS One 6(4):e19537

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gaj T, Barbas CF III (2014) Genome engineering with custom recombinases. Methods Enzymol 546:79–91

    Article  CAS  PubMed  Google Scholar 

  9. Prorocic MM, Wenlong D, Olorunniji FJ, Akopian A, Schloetel J-G, Hannigan A, McPherson AL, Stark WM (2011) Zinc-finger recombinase activities in vitro. Nucleic Acids Res 39:9316–9328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Arnold PH, Blake DG, Grindley NDF, Boocock MR, Stark WM (1999) Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity. EMBO J 18:1407–1414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89

    Article  CAS  PubMed  Google Scholar 

  12. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 1599–1615

    Google Scholar 

  13. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  14. Laniel M-A, Béliveau A, Guérin SL (2001) Electrophoretic mobility shift assays for the analysis of DNA-protein interactions. In: Moss T (ed) DNA-protein interactions, Principles and protocols. Methods in molecular biology, vol 148. Humana Press, Totowa, pp 13–30

    Chapter  Google Scholar 

  15. Olorunniji FJ, He J, Wenwieser SVCT, Boocock MR, Stark WM (2008) Synapsis and catalysis by activated Tn3 resolvase mutants. Nucleic Acids Res 36:7181–7191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgment

We are very grateful to Miss Arlene McPherson for technical support. This work was supported by the BBSRC sLOLA grant BB/003356/1to F.J.O., S.J.R., and W.M.S.

Author information

Authors and Affiliations

  1. Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow, G12 8QQ, Scotland

    Femi J. Olorunniji & W. Marshall Stark

  2. SynthSys—Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, CH Waddington Building, The King’s Buildings, Mayfield Road, Edinburgh, EH9 3JD, Scotland

    Susan J. Rosser

Authors
  1. Femi J. Olorunniji
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan J. Rosser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. Marshall Stark
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to W. Marshall Stark .

Editor information

Editors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Nikolai Eroshenko

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Olorunniji, F.J., Rosser, S.J., Marshall Stark, W. (2017). Purification and In Vitro Characterization of Zinc Finger Recombinases. In: Eroshenko, N. (eds) Site-Specific Recombinases. Methods in Molecular Biology, vol 1642. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7169-5_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7169-5_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7167-1

  • Online ISBN: 978-1-4939-7169-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation