Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies.
Two-hybrid methods detect interactions between two proteins fused at the C-termini of, respectively, a DNA-binding domain and the activation domain of a transcriptional activator. Thus the N-terminus of none of these proteins is available for interaction. We have tested whether a bait protein with a reverted polarity (i.e. N-bait-LexA-C) is suitable for two-hybrid interaction. We show that such constructs give a specific interaction signal, and document two cases where the sensitivity is dramatically increased. Such constructs might lead to the identification of partners missed during classical two-hybrid screens.
Béranger
F
F
INSERM U-248, Institut Curie, 26 rue d'Ulm, 75231 Paris cedex 05, France.
Aresta
S
S
de Gunzburg
J
J
Camonis
J
J
D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry; D005656:Fungal Proteins / Q000096:biosynthesis; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000096:biosynthesis*; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000096:biosynthesis; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae