pBridge酵母三杂交系统载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pBridge酵母三杂交系统载体 BioVector NTCC质粒载体菌种细胞基因保藏中心 pBridge expresses two proteins: a DNA-binding domain fusion, and an additional protein (1–3). pBridge thus allows establishment of three-hybrid systems when used in combination with an activation domain fusion vector and yeast strains from any of Clontech's GAL4-based two-hybrid systems, including Matchmaker™ Gold. This vector generates a hybrid protein that contains the sequences for the GAL4 DNA-binding domain (DNA-BD; a.a. 1–147) and the sequence cloned into MCS I. The fusion protein is expressed in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (NLS) that are an intrinsic part of the GAL4 DNA-BD (3). An additional gene of interest can be cloned into MCS II which is located downstream of an HA epitope and a second NLS. The resulting fusion protein is conditionally expressed from the Met25 promoter in response to methionine levels in the medium; i.e., it is repressed in the presence of 1 mM methionine and expressed in the absence of methionine (1).pBridge is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae. It carries the bla gene (for ampicillin resistance in E. coli) and the TRP1 nutritional marker that allow yeast auxotrophs carrying pBridge to grow on limiting synthetic medium lacking tryptophan. Note: Yeast strain Y187 is a methionine auxotroph; therefore, haploid Y187 harboring pBridge cannot be grown on medium lacking methionine. Use: The pBridge Vector now makes it possible to investigate ternary protein complex formation (1, 2). pBridge contains two distinct multiple cloning sites to allow expression of the DNA-BD fusion as well as a third protein. When pBridge is used in conjunction with the AD fusion vector and yeast strains from any one of Clontech’s GAL4-based two-hybrid systems, a ‘three-hybrid’ system can be established hat is dependent on the expression of a third protein. The yeast two-hybrid system has proven to be a powerful molecular approach for detecting protein-protein (binary) interactions and has contributed significantly to the dissection of many molecular pathways. However, the two-hybrid system is designed to detect the interaction between just two proteins, which are expressed as the AD and BD fusions. The figure below demonstrates the more complex protein interactions that can be investigated with the three-hybrid system.The third protein in this system can participate in the interaction in several ways: as a “bridge”, interacting with two proteins that do not directly interact with each other; to stabilize a weak interaction between two proteins; or as an inhibitor or modifier (e.g., kinase; 3) of one or both of the proteins. Alternatively, a competitor of a two-hybrid interaction can be expressed from this promoter to confirm the specificity of the two-hybrid interaction (1). The conditional expression of the third protein allows investigation of its role in the interaction between the AD and BD fusion proteins. Like the two-hybrid system, the three-hybrid system can be used to screen libraries to clone new interacting partners, either for the third protein or the binding domain fusion. Expression of the third protein is controlled by a conditional methionine promoter (PMet25) such that it is expressed in the absence of methionine. This allows expression to be switched on or off by a simple replica plating step. The effect of the third protein is indicated by expression of a reporter or nutritional marker. To facilitate experiments with pBridge, we offer three drop-out media supplements that lack methionine: –His/–Leu/–Met/–Trp (Cat No. 630429); –Leu–Met/–Trp (Cat. No. 630430); and –Met/–Trp. Location of features: • Promoter Fragment containing the S. cerevisiae ADH1 promoter: 10–406 • GAL4 DNA-binding domain polypeptide Start codon: 434–436; stop codon: 953–955 GAL4 codons 1–147: 434–874 • MCS I: 878–905 • Transcription termination signal Fragment carrying the S. cerevisiae ADH1 terminator: 921–1112 • TRP1 coding sequence Start codon: 1835–1833; stop codon: 1163–1161 • S. cerevisiae MET25 promoter: 2117–2609 • HA epitope and nuclear localization sequence: 2610–2699 • MCS II: 2700–2723 • S. cerevisiae PGK terminator: 2733–3120 • Col E1 origin of replication: 3324–3967 • Ampicillin resistance gene (β-lactamase): 5045–3968 Promoter: 5045–5017 Coding sequence: 4975–4115 • Fragment containing the 2 μ origin of replication: 5362–6526 Primer locations: • Matchmaker DNA-BD 5' Insert Screening Amplimer (Cat. No. 5417-1) or GAL4 BD Sequencing Primer (Cat. No. 6474-1): 827––843 • Matchmaker DNA-BD 3' Insert Screening Amplimer (Cat. No. 5417-1): 1015–994 Propagation in E. coli: • Suitable host strains: DH5α and other general purpose strains. • Selectable marker: plasmid confers resistance to ampicillin (50 μg/ml) to E. coli hosts. • E. coli replication origin: Col E1 • Copy number: 15–20 Propagation in S. cerevisiae: • Suitable host strains: Y187(α)*, Y190(a), HF7c(a), CG1945(a), PJ69-2A(a), PJ69-4A(a)*, SFY526(a), AH109(a) and Y2H Gold. Note: When using pBridge, yeast strains need to be streaked 3 times to single colony on agar plates lacking Methionine (SD/-Met) since they tend to lose tolerance to this medium if not maintained on it routinely. • Selectable marker: TRP1 • S. cerevisiae replication origin: 2 μ • Copy number: multiple copy References: 1. Tirode, F., et al. (1997) J. Biol. Chem. 272:22995–22999. 2. Brachmann, R. & Boeke, J. (1997) Curr. Opin. Biotechnol. 8:561–568. 3. Osborne, M., et al. (1995) Biotechnology 13:1474–1478. * These strains are methionine auxotrophs; protein cannot be expressed from MCS II of pBridge in these strains. However, these strains may be used when mating to a methionine autotroph. b [Supplier来源] http://www.biovector.net