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Detecting protein-protein interactions with GFP-fragment reassembly

Nature Methods volume 1, pages 255–262 (2004)Cite this article

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The detection of protein-protein interactions in vivo is of critical importance to our understanding of biological processes. The classic library approach has been to use the yeast two-hybrid screen, where an interaction between known bait and unknown prey proteins leads to restoration of transcription factor activity1. However, its use is limited by host organism and nuclear localization requirements, and a tendency to detect indirect interactions (false positives). Bacterial two-hybrid screens have eliminated localization requirements and simplified many technical aspects of the procedure2. An innovative approach has been the reassembly of protein fragments, which then directly report interactions. A suitable reporter protein is dissected at the genetic level, and the fragments are fused to bait and prey, which are then coexpressed in vivo. Bait and prey interaction brings the reporter fragments together, facilitating reassembly of the active reporter protein, giving a direct readout of the association. This method has been demonstrated for dihydrofolate reductase3,4, ubiquitin5 and the green fluorescent protein6 (GFP) from Aequorea victoria. We recently described improvements to the original screen based on the reassembly of the GFP enhanced-stability mutant sg100 in Escherichia coli7. Our system, presented in the protocol that follows, consists of two plasmid vectors for the independent expression of fusions with N- and C-terminal fragments of GFP, and allows for simple visual detection of protein-protein interactions with a KD as weak as 1 mM.

  • Plasmid vectors pET11a-link-NGFP and pMRBAD-link-CGFP and positive control vectors pET11a-Z-NGFP and pMRBAD-Z-CGFP are available upon request (Fig. 1)

    Figure 1: GFP-fragment reassembly vectors and cloning linker sequences.
    figure 1

    (a) Oligonucleotide linker sequences for amplification of target bait and prey DNA. NGFP in-frame fusions are made through the XhoI site in the forward primer sequence. The reverse primer should contain a stop codon followed by a BamHI site. Cloning into pMRBAD-link-CFGP requires a forward primer in frame with the ATG in the NcoI site. Fusion to CGFP is achieved through the in-frame AatII site primer sequence. The symbol (xxx) represents codons of the template gene to be amplified and cloned into the vectors. Six codons (18 bp) are usually sufficient to obtain specific amplification of most target sequences. The six-base extension (5′-aataat) is required for efficient cutting by restriction enzymes. (b) Cloning linker sites and key features of the GFP-fragment vectors. pET11a-link-GFP contains the ampicillin resistance marker (ApR) and ColE1 origin of replication (ori). The XmaI site in the linker is lost during cloning; thus, digestion with XmaI can be used to eliminate the background that results from religation. Fusion expression is achieved in a BL21 DE3 host with the addition of IPTG, which results in transcription from the T7 promoter (PT7). The rop gene product regulates the copy number of plasmids containing the ColE1 origin of replication, and the lacI gene product, the Lac repressor, reduces leaky expression in DE3 bacterial hosts. pMRBAD-link-GFP contains the kanamycin resistance gene (KnR) and p15A origin of replication. The linker SphI site is lost during cloning; thus, digestion with SphI can be used to eliminate the background that results from religation. Fusion expression is under the control of the PBAD promoter (regulated by the product of the araC gene) and is induced with L-(+)-arabinose. Note that the resulting GFP fusions have different orientations relative to the GFP fragment, which may affect the likelihood of successful GFP reassembly. Sequence details of these vectors can be found in Supplementary Note online and at http://www.csb.yale.edu/people/regan/publications.html. The structures of the vectors can be found in Supplementary Fig. 1 online.

    Full size image

  • Primers for cloning into pET11a-link-NGFP and pMRBAD-link-CGFP (Fig. 1)

  • Selective media: LB containing 100 μg/ml ampicillin and LB containing 35 μg/ml kanamycin

  • Restriction enzymes: NcoI, BamHI, AatII, XhoI, XmaI and SphI (New England Biolabs (NEB))

  • Thermostable DNA polymerase (for example, Deep Vent Polymerase, NEB)

  • dNTP solution (10 mM)

  • Calf intestinal alkaline phosphatase (NEB)

  • T4 DNA ligase (NEB)

  • E. coli strains DH10B and BL21 (DE3), prepared as competent cells (preferably electrocompetent)

  • Sequencing primers for pET11a-link-NGFP (T7 terminator primer 5′-tatgctagttattgctcag-3′) and pMRBAD-link-CGFP (5′-ctactgtttctccatacccg-3′)

  • ExoSAP-IT (USB)

  • Screening medium: for 250 ml LB agar (for about ten plates) add 250 μl of 10 mM IPTG, 2.5 ml of 20% arabinose, 250 μl of 100 mg/ml ampicillin (100 μg/ml) and 250 μl of 35 mg/ml kanamycin (35 μg/ml). Sterilize all additives by passing through a 0.2 μm filter.

  • Lysis buffer: 50 mM Tris HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 1 mg/ml lysozyme, 0.5 mM CaCl2, 5 mM MgCl2

  • Nucleases: DNase and RNase (Sigma)

  • Ni-NTA agarose (Qiagen)

  • Wash buffer: 50 mM Tris HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole

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Figure 1: GFP-fragment reassembly vectors and cloning linker sequences.
Figure 2: (a) Schematic plate layout for screening interactions with the GFP reassembly screen.

References

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Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, New Haven, 06520-8114, Connecticut, USA

    Christopher GM Wilson, Thomas J Magliery & Lynne Regan

  2. Department of Chemistry, Yale University, P.O. Box 208107, New Haven, 06520-8107, Connecticut, USA

    Lynne Regan

Authors
  1. Christopher GM Wilson
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  2. Thomas J Magliery
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Supplementary information

Supplementary Fig. 1

Vector maps of pET11a-link-NGF and pMRBAD-link-CGFP. (PDF 104 kb)

Supplementary Note

Sequences of the plasmid vectors pET11a-link-NGF and pMRBAD-link-CGFP. (PDF 85 kb)

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Wilson, C., Magliery, T. & Regan, L. Detecting protein-protein interactions with GFP-fragment reassembly. Nat Methods 1, 255–262 (2004). https://doi.org/10.1038/nmeth1204-255

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