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We have adapted the basic two-hybrid strategy to create a novel Dual
Bait system, where bait 1 is fused to LexA DNA-binding-domain, and bait
2 is fused to lambda cI DNA-binding-domain. This system allows single-step
screening of libraries for two sets of positives: one set of positives
can be registered through lexAop activation of LEU2 and LacZ,
and a second set through cIop activation of LYS2 and GusA, and both negatively
controlled against each other. Using the selective interactions of a set
of control proteins of predetermined interaction specificity (Ras and Krev-1
with Raf, Krit1, and RalGDS) we have demonstrated that Dual Bait system
allows effective detection of specific interactions. We have further shown
that in a cDNA library screen with two separate baits, we were able
to reduce the number of false positives isolated in a two-hybrid
screen with a difficult bait. Finally, as a demonstration of the utility
of the Dual Bait in mutational analysis of protein-specific interaction
domains, we screened a library of mutants of Pak1 (p21-activated kinase),
which normally associates with both activated Cdc42 and Rac1, to identify
a series of Pak1 mutants which selectively bind to only one or the other
of these GTPases. We anticipate that Dual Bait reagents will prove useful
in genomic applications, and in high-throughput specific protein-drug screening efforts.
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| Original Paper describing creation and initial set of reagents for Dual Bait | Dual Bait was adapted by Invitrogen to create Dual Bait Hybrid Hunter | Extended set of modified and improved Dual Bait reagents | Protocol on using Dual Bait:
under construction! |
| Serebriiskii, I., Khazak, V., and Golemis, E. A.
A two-hybrid dual bait system to discriminate specificity of protein interactions. |
PDF downloads:
Expression Newsletter, see pp.10-11; Catalog Entry, see p.12 |
Manuscript in preparation. Consult the table and contact us for more detailed information | Serebriiskii, I., Mitina, O., Chernoff, J. , and E.A.Golemis. Use of a two-hybrid dual bait system to discriminate specificity of protein interactions in small GTPases. In "Methods in Enzymology: Ras Regulators and Effectors", ed. C.J. Der. Academic Press, 2000. |