Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.
Yeast two-hybrid screens often produce vastly non-overlapping interaction data when the screens are conducted in different laboratories, or use different vectors, strains, or reporter genes. Here we investigate the underlying reasons for such inconsistencies and compare the effect of seven different vectors and their yeast two-hybrid interactions. Genome-wide array screens with 49 motility-related baits from Treponema pallidum yielded 77 and 165 interactions with bait vectors pLP-GBKT7 and pAS1-LP, respectively, including 21 overlapping interactions. In addition, 90 motility-related proteins from Escherichia coli were tested in all pairwise combinations and yielded 140 interactions when tested with pGBKT7g/pGADT7g vectors but only 47 when tested with pDEST32/pDEST22. We discuss the factors that determine these effects, including copy number, the nature of the fusion protein, and species-specific differences that explain non-conserved interactions among species. The pDEST22/pDEST32 vectors produce a higher fraction of interactions that are conserved and that are biologically relevant when compared with the pGBKT7/pGADT7-related vectors, but the latter appear to be more sensitive and thus detect more interactions overall.
Rajagopala
Seesandra Venkatappa
SV
J Craig Venter Institute, Rockville, MD 20850, USA.
Hughes
Kelly T
KT
Uetz
Peter
P
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D020409:Molecular Motor Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014210:Treponema pallidum / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D020409:Molecular Motor Proteins