Two-hybrid, protein interactions

Figure 5.2. High-throughput mating assay for two-hybrid protein interaction screening. Yeast strains containing individual bait and prey clones are combined in a well and allowed to mate. Diploids are then selected and scored for a protein-protein interaction using the selection provided by the transcriptional reporter gene.

For a complete list of commercially available destination vectors, please visit uniw.in-vitrogen.com. These cover a broad array of applications including protein expression in E. coli, yeast, insect, and mammalian cells, yeast two hybrid protein interaction, and gene knock-down RNAi vectors. 

Two-hybrid protein interaction technology for target identification... 

Two-hybrid protein interaction technology enables isolation and characterization of potential drug targets by identifying specific proteins that bind with other peptides or proteins that are known to be part of a signaling pathway. This protein interaction technology is directed at ... 

A Two-Hybrid Protein interaction System to identify Factors That interact with Retinoid and Vitamin D Receptors... 

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

Ehlert, A., Weltmeier, F., Wang, X., et al. (2006). Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts establishment of a heterodimeiization map of group C and group S bZIP transcription factors. Plant J., 46, 890-900. 

Fig. 8.1 The yeast two-hybrid system. Two hybrid proteins are expressed in yeast. The "bait" protein consists of protein X fused to the DNA-binding domain of the GAL4 transcription factor (BD). The "prey" protein consists of protein Y fused to the GAL4 transcriptional activation domain (AD). Neither hybrid alone is able to activate transcription of the reporter gene (p-galactosi-dase in this example). However, if a protein-protein interaction occurs between protein X and protein Y, a functional transcriptional activator is generated resulting in expression of p-galactosidase which is detected as a color change phenotype of the host yeast cells growing in the presence of a suitable substrate (e.g. X-gal).

The Two-Hybrid System to Identify Proteins Involved in Specific Protein-Protein Interactions... 

Chien, C.-X, et al., 1991. The two-hybrid system A method to identify and clone genes for proteins that interact with a protein of interest. Proceedings of the National Academy of Sciences U.S.A. 88 9578 — 9582. 

PIAS (protein inhibitors of activated STATs) proteins were first discovered in yeast-two-hybrid screens as interacting molecules with STAT transcription factors. The mammalian family consists ofthe founding member PIAS3, which was described as a repressor of STAT3, and three additional members, PIAS1, PIASy (also known as PIAS4), and PIASx (also known as... 

Two-hybrid analysis protein-protein interactions in viral systems... 

Two-hybrid analysis ofprotein-protein interactions in bacteria... 

The genome of the Helicobacterpylori bacterium is 1.6 million base pairs in size and encodes 1590 ORFs (Tomb et al., 1997). The comprehensive two-hybrid library screen performed with these ORFs differs from the yeast experiments described above in that the Gal4 activation domain library used consisted of over ten million random genomic fragments (Rain et al., 2001). Thus, the potential problem of full-size ORFs masking protein-protein interactions is reduced. A total of 261 ORFs were fused to the Gal4 DNA binding domain to create a set of baits. These ORFs... 

Two-hybrid analysis of protein-protein interactions in Caenorhabditis elegans... 

Bacterial two-hybrid system for the detection of protein-protein interactions... 

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

Figure 5.5. A. Schematic illustration of the E. coli RNA polymerase showing the domain structure of the a subunit. The cx-NTD domain is responsible for assembly of RNAP while the a-CTD domain binds DNA and is a target for transcriptional activators. B. The two-hybrid system is based on the interaction of proteins that are fused to the X repressor and NTD domain of the a subunit of RNAP. In the example shown, Gal4 interacts with Gall IP to recruit RNAP to the promoter and activate transcription of the lacZ reporter gene. Figure adapted from Dove and Hochschild (1998).

The bacterial one and two-hybrid systems have potential advantages over the yeast two-hybrid system due to the higher transformation efficiency and faster growth rate of coli. To date, however, the bacterial two-hybrid system has not been used for genome-scale analysis of protein-protein interactions. 

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

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