Detecting protein-protein interactions with GFP-fragment reassembly

Abstract

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 activity 1. 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 procedure 2. 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 reductase 3,4 , ubiquitin 5 and the green fluorescent protein6 (GFP) from Aequorea victoria. We recently described improvements to the original screen based on the reassembly of the GFP enhancedstability mutant sg100 in Escherichia coli 7. 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 K D as weak as 1 mM. MATERIALS REAGENTS 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) 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 CaCl 2 , 5 mM MgCl 2 Nucleases: DNase and RNase (Sigma) Ni-NTA agarose (Qiagen) Wash buffer: 50 mM Tris HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole EQUIPMENT Thermal cycler programmed with the desired amplification protocol Handheld long-wave UV lamp (365 nm) Disposable standing column (Bio-Ra...

Histidine-containing Phosphotransfer Protein-B (HptB) Regulates Swarming Motility through Partner-switching System in Pseudomonas aeruginosa PAO1 Strain

“…BiFC Assay-The BiFC assay was performed as described previously (26,27) with slight modifications. The positive control plasmid pair was pET11a-Z-NGFP and pMRBAD-Z-CGFP encoding the green fluorescence protein fused with one of the antiparallel leucine zipper tags, respectively.…”

Recent progress in the construction methodology of fluorescent biosensors based on biomolecules

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The domain structure of Entamoeba α-actinin2

“…The plates were incubated at room temperature (20ºC) for 24 h. Single colonies were then picked and streaked on screening LB plates containing 100 μg/ml carbencillin, 35 μg/ml kanamycin and 10 μM IPTG and 0.2% (w/v) arabinose. The cells were grown at for 16 h at 30ºC followed by 1-3 days at 25ºC [38].…”