Turn Up the Lex Appeal

Schematic illustration of the LexA-based yeast two-hybrid interaction. Source: OriGene Technologies, Inc. 97/98 Catalog. Reprinted with permission. The DupLEX-ATM system from OriGene Technologies is a LexA-based two-hybrid assay for the detection of protein-protein interactions in vivo (J. Gyuris et al., Cell 75:791-803, 1993). The LexA system is conceptually similar to the prototypical GAL4-based two-hybrid system, both of which are based on the bipartate nature of yeast transcriptional activators. These protein complexes can be used to activate transcription when their two domains (DNA binding and transcriptional activator domains) are brought into proximity. In two-hybrid systems, this is mediated by the presence of interacting proteins that have been fused to one of the two domains on separate vectors--the so-called bait vector-- which contains the DNA-binding (LexA) domain, and the target vector, which contains the transcriptional activator, (B42) domain (S. Fields and O. Song, Nature, 340:245-247, 1989; C.T. Chien et al., Proceedings of the National Academy of Sciences USA, 88:9578-82, 1991). In OriGene's Duplex-A Yeast Two-Hybrid system, the protein of interest ("Bait") is expressed as a fusion protein with LexA, an E. coli DNA-binding protein. The candidate binding partner ("Target") can be a test protein of choice or a mixture of proteins generated by an expression library. In the latter case, this method can be used to discover previously unknown binding proteins. The Target is fused at the C terminal to the transcriptional activation domain of an E. coli protein (B42) and a hemagglutinin (HA) epitope tag. The HA epitope tag is useful for the detection and purification of the fusion protein via immunochemical techniques. Neither LexA bound upstream of a reporter gene nor B42 alone can activate transcription of the reporter. If the two are brought together via fusions with the Bait and Target proteins, transactivation occurs, and the reporter gene is turned on. The LexA-based yeast two-hybrid systems utilize two different reporter genes: the plasmid-borne lacZ and LEU2, which is integrated in the yeast host strain chromosome. Galactose can be used to induce the expression of the B42-HA-Target fusion protein, which is under the control of a GAL1 promoter. Having inducible expression of the Target fusion proteins is a benefit when working with potentially toxic proteins, as it decreases exposure of the host yeast strain to the foreign proteins. While the LexA-Bait constructs supplied with the kit are not under the control of the GAL1 promoter, if inducible expression is desired, OriGene recommends pGilda, a low-copy-number plasmid that places expression of the Bait under control of the GAL1 promoter. BAIT/TYPE OF PROTEINPREY/TYPE OF PROTEINMEANS OF DETECTING INTERACTION Insulin receptor/receptorIRS-1/unknownpredefined set of proteins C-abl domain/protein kinaseCrk/adaptor proteinlibrary screen TGF-ß R-I/receptorTGF-ß R-II/receptorlibrary screen DmCdc2/cell division kinaseCdi 3,7/cyclinpredefined set of proteins This data was taken from the web site of Erica Golemis at the Fox Chase Cancer Center (Philadelphia, Pa.). A more complete table can be found at: www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html "The kit is complete with all necessary control vectors and strains," says Dr. Luca Pellegrini, a visiting fellow at the National Institutes of Health who has successfully used OriGene's Duplex-A Yeast Two-Hybrid system. In fact, OriGene's LexA-based system contains some components that are not available from other vendors. For example, most commercially available kits are supplied with a yeast strain with six LexA-binding sites upstream of the LEU2 reporter gene. OriGene's kit includes additional yeast strains not generally available from other vendors that have different numbers of LexA-binding sites. The most sensitive strain contains six binding sites for LexA and may be able to detect interactions of proteins with low binding affinities. However, some Bait proteins will, in the absence of Target, activate the reporter gene. This "autoactivation" activity can sometimes be alleviated by switching to one of the strains with fewer binding sites. "From what I have heard, using a less sensitive bait has about a 50 percent chance of overcoming the autoactivation problem for a particular bait," says Dr. Michael R. Murphy, scientific operations manager for OriGene Technologies, Inc. A fourth yeast strain, which contains no LexA-binding sites, is included as a negative control. The other reporter, lacZ, is encoded on a high-copy-number plasmid that enables one to assay ß-galactosidase directly on the culture plate. As is the case with the LEU2 reporter, a panel of lacZ constructs of varying sensitivities is included. OriGene also markets an extensive line of premade cDNA libraries designed to be used with this system. "The cDNA library turned out to be quite good," says Dr. Pellegrini, who purchased a human liver cDNA library along with the Duplex-A Yeast Two-Hybrid system. Adds Dr. Pellegrini, "We screened 5 million clones and [obtained] three different interacting proteins.... The expression level for each of the three genes is fairly good. The size of the inserts were from 1.1 to 2Kb. Overall, I definitively recommend Origene's cDNA library...." For more information please visit OriGene's web site at www.origene.com. Or call (301) 340-3188, or (888) 267-4436. The author, Deborah Wilkinson, can be contacted at deborahwilkinson@sprynet.com.

Turn Up the Lex Appeal

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