Skip to main content

High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

The identification of soluble, folded domains of proteins is a recurring task in modern molecular biology. We detail a protocol for identifying compact soluble protein domains using a self-assembling two-part split-GFP comprised of a detector fragment (GFP β-strands 1 through 10, or GFP1–10) and a tagging fragment (GFP β-strand 11, or GFP11). The assay is performed in E. coli cells and in cell extracts. A selection step insures the protein fragments are in frame and contain no stop codons, while an inverse PCR is used to enrich protein fragment libraries containing a specific target sequence.

Key words

  • Split-GFP
  • Domain trapping
  • Protein solubility
  • Protein tagging
  • Protein fragment complementation

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-9624-7_15
  • Chapter length: 13 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   209.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-9624-7
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   269.00
Price excludes VAT (USA)
Hardcover Book
USD   379.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Cabantous S, Terwilliger T, Waldo G (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol 23:102–107. https://doi.org/10.1038/nbt1044

    CAS  CrossRef  PubMed  Google Scholar 

  2. Pedelacq JD, Nguyen HB, Cabantous S, Mark BL, Listwan P, Bell C, Friedland N, Lockard M, Faille A, Mourey L, Terwilliger TC, Waldo GS (2011) Experimental mapping of soluble protein domains using a hierarchical approach. Nucleic Acids Res 39:e125. https://doi.org/10.1093/nar/gkr548

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  3. Smith VF, Matthews CR (2001) Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E. coli: fragment complementation and circular permutation reveal stable, alternatively folded forms. Protein Sci 10:116–128. https://doi.org/10.1110/ps.26601

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Cabantous S, Waldo GS (2006) In vivo and in vitro protein solubility assays using split GFP. Nat Methods 3:845–854. https://doi.org/10.1038/nmeth932

    CAS  CrossRef  Google Scholar 

  5. Blommel PG, Martin PA, Wrobel RL, Steffen E, Fox BG (2006) High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. Protein Expr Purif 47:562–570. https://doi.org/10.1016/j.pep.2005.11.007

    CAS  CrossRef  Google Scholar 

  6. Hoskins RA, Stapleton M, George RA, Yu C, Wan KH, Carlson JW, Celniker SE (2005) Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP). Nucleic Acids Res 33. https://doi.org/10.1093/nar/gni184

    CrossRef  Google Scholar 

  7. Faille A, Gavalda S, Slama N, Lherbet C, Maveyraud L, Guillet V, Laval F, Quémard A, Mourey L, Pedelacq JD (2017) Insights into substrate modification by dehydratases from Type I polyketide synthases. J Mol Biol 429:1554–1569. https://doi.org/10.1016/j.jmb.2017.03.026

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jean-Denis Pedelacq .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Massemin, A., Cabantous, S., Waldo, G.S., Pedelacq, JD. (2019). High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

  • eBook Packages: Springer Protocols