This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. A major disadvantage of the two-hybrid system is the number of technical false-positive interactions that can arise. These false-positives represent activation of the HIS3 reporter gene by mechanisms that do not involve a direct physical interaction of bait and prey proteins. In an effort to diminish these false positives, we have been working on incorporating a second reporter system that relies on a read-out derived from a direct interaction of bait and prey proteins. We split the firefly luciferase gene into two halves (amino acid residues 1-398 and 395-550) and incorporated them into the existing two-hybrid system. When a bait and prey protein bind one another, the two halves of luciferase are brought into close proximity with one another and are able to re-associate and generate luminescence in the presence of D-luciferin substrate. Early tests revealed that the two halves of luciferase could self-associate even in the absence of an interacting bait and prey protein pair. This self-association led to both luminescence and growth on -HIS plates. The presence of an interacting bait and prey pair led to further luminescence and stronger growth on -HIS plates, but the signal to noise ratio was only about 1.5 to 2-fold when comparing an interacting pair versus a protein with empty prey vector. Thus we randomly mutagenized the two halves of luciferase in an effort to abolish this self-association. The mutagenesis screen revealed several mutants with decreased levels of self-association but also decreased luminescence and growth on -HIS with an interacting protein pair. The screen also revealed several mutations that led to an overall increase in levels of luminescence. Therefore, we coupled these two features, low self-association of luciferase and higher luminescence, with the best combination being a change of aspartic acid residue 375 to a glutamic acid residue (decreased self-association) and a change of leucine residue 530 to an arginine residue (increased luminescence). This combination of mutations led to a 4- to 6-fold difference in signal to noise with an interacting pair vs. an empty vector. We are currently testing this dual reporter system in a library screen setting to determine whether it can distinguish real interactions from false positives.
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