A combined yeast/bacteria two-hybrid system: development and evaluation.
Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.
Serebriiskii
Ilya G
IG
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. ig_serebriiskii@fccc.edu
Fang
Rui
R
Latypova
Ekaterina
E
Hopkins
Richard
R
Vinson
Charles
C
Joung
J Keith
JK
Golemis
Erica A
EA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D025941:Protein Interaction Mapping; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf