pBridgeTM 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 (#K1604-1, #K1605-1, #K1612-1). 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.

載體應(yīng)用

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, the MATCHMAKER Two-Hybrid System (#K1605-1) or the MATCHMAKER Two-Hybrid System 2 & 3 (#K1604-1 & #K1612-1), a ‘three-hybrid’ system can be established that 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. CLONTECH’s MATCHMAKER GAL4- and LexA-based two-hybrid systems and cDNA libraries have facilitated the widespread use of this technology. 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 (#8620-1); –Leu–Met/–Trp (#8621-1); and –Met/–Trp (#8622-1).

0001 GCTTGCATGC AACTTCTTTT CTTTTTTTTT CTTTTCTCTC TCCCCCGTTG TTGTCTCACC 
0061 ATATCCGCAA TGACAAAAAA ATGATGGAAG ACACTAAAGG AAAAAATTAA CGACAAAGAC 
0121 AGCACCAACA GATGTCGTTG TTCCAGAGCT GATGAGGGGT ATCTCGAAGC ACACGAAACT 
0181 TTTTCCTTCC TTCATTCACG CACACTACTC TCTAATGAGC AACGGTATAC GGCCTTCCTT 
0241 CCAGTTACTT GAATTTGAAA TAAAAAAAAG TTTGCTGTCT TGCTATCAAG TATAAATAGA 
0301 CCTGCAATTA TTAATCTTTT GTTTCCTCGT CATTGTTCTC GTTCCCTTTC TTCCTTGTTT 
0361 CTTTTTCTGC ACAATATTTC AAGCTATACC AAGCATACAA TCAACTCCAA GCTTGAAGCA 
0421 AGCCTCCTGA AAGATGAAGC TACTGTCTTC TATCGAACAA GCATGCGATA TTTGCCGACT 
0481 TAAAAAGCTC AAGTGCTCCA AAGAAAAACC GAAGTGCGCC AAGTGTCTGA AGAACAACTG 
0541 GGAGTGTCGC TACTCTCCCA AAACCAAAAG GTCTCCGCTG ACTAGGGCAC ATCTGACAGA 
0601 AGTGGAATCA AGGCTAGAAA GACTGGAACA GCTATTTCTA CTGATTTTTC CTCGAGAAGA 
0661 CCTTGACATG ATTTTGAAAA TGGATTCTTT ACAGGATATA AAAGCATTGT TAACAGGATT 
0721 ATTTGTACAA GATAATGTGA ATAAAGATGC CGTCACAGAT AGATTGGCTT CAGTGGAGAC 
0781 TGATATGCCT CTAACATTGA GACAGCATAG AATAAGTGCG ACATCATCAT CGGAAGAGAG 
0841 TAGTAACAAA GGTCAAAGAC AGTTGACTGT ATCGCCGGAA TTCCCGGGGA TCCGTCGACC 
0901 TGCAGCCAAG CTAATTCCGG GCGAATTTCT TATGATTTAT GATTTTTATT ATTAAATAAG 
0961 TTATAAAAAA AATAAGTGTA TACAAATTTT AAAGTGACTC TTAGGTTTTA AAACGAAAAT 
1021 TCTTGTTCTT GAGTAACTCT TTCCTGTAGG TCAGGTTGCT TTCTCAGGTA TAGCATGAGG 
1081 TCGCTCTTAT TGACCACACC TCTACCGGCA TGCCGGCAAG TGCACAAACA ATACTTAAAT 
1141 AAATACTACT CAGTAATAAC CTATTTCTTA GCATTTTTGA CGAAATTTGC TATTTTGTTA 
1201 GAGTCTTTTA CACCATTTGT CTCCACACCT CCGCTTACAT CAACACCAAT AACGCCATTT 
1261 AATCTAAGCG CATCACCAAC ATTTTCTGGC GTCAGTCCAC CAGCTAACAT AAAATGTAAG 
1321 CTTTCGGGGC TCTCTTGCCT TCCAACCCAG TCAGAAATCG AGTTCCAATC CAAAAGTTCA 
1381 CCTGTCCCAC CTGCTTCTGA ATCAAACAAG GGAATAAACG AATGAGGTTT CTGTGAAGCT 
1441 GCACTGAGTA GTATGTTGCA GTCTTTTGGA AATACGAGTC TTTTAATAAC TGGCAAACCG 
1501 AGGAACTCTT GGTATTCTTG CCACGACTCA TCTCCATGCA GTTGGACGAT ATCAATGCCG 
1561 TAATCATTGA CCAGAGCCAA AACATCCTCC TTAGGTTGAT TACGAAACAC GCCAACCAAG 
1621 TATTTCGGAG TGCCTGAACT ATTTTTATAT GCTTTTACAA GACTTGAAAT TTTCCTTGCA 
1681 ATAACCGGGT CAATTGTTCT CTTTCTATTG GGCACACATA TAATACCCAG CAAGTCAGCA 
1741 TCGGAATCTA GAGCACATTC TGCGGCCTCT GTGCTCTGCA AGCCGCAAAC TTTCACCAAT 
1801 GGACCAGAAC TACCTGTGAA ATTAATAACA GACATACTCC AAGCTGCCTT TGTGTGCTTA 
1861 ATCACGTATA CTCACGTGCT CAATAGTCAC CAATGCCCTC CCTCTTGGCC CTCTCCTTTT 
1921 CTTTTTTCGA CCGAATTAAT TCGTAATCAT GTCATAGCTG TTTCCTGTGT GAAATTGTTA 
1981 TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC 
2041 CTAATGAGTG AGGTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG 
2101 AAACCTGTCG TGCCAGGAAG ATCCGAGGCC TAGCTTCTAA TTCTTCCAAC ATACAATGGG 
2161 AGTTTGGCCG AGTGGTTTAA GGCGTCAGAT TTAGGTGGAT TTAACCTCTA AAATCTCTGA 
2221 TATCTTCGGA TGCAAGGGTT CGAATCCCTT AGCTCTCATT ATTTTTTGCT TTTTCTCTTG 
2281 AGGTCACATG ATCGCAAAAT GGCAAATGGC ACGTGAAGCT GTCGATATTG GGGAACTGTG 
2341 GTGGTTGGCA AATGACTAAT TAAGTTAGTC AAGGCGCCAT CCTCATGAAA ACTGTGTAAC 
2401 ATAATAACCG AAGTGTCGAA AAGGTGGCAC CTTGTCCAAT TGAACACGCT CGATGAAAAA 
2461 AATAAGATAT ATATAAGGTT AAGTAAAGCG TCTGTTAGAA AGGAAGTTTT TCCTTTTTCT 
2521 TGCTCTCTTG TCTTTTCATC TACTATTTCC TTCGTGTAAT ACAGGGTCGT CAGATACATA 
2581 GATACAATTC TATTACCCCC ATCCATACAA TGGGCCATAT GGCTTCTAGC TATCCTTATG 
2641 ACGTGCCTGA CTATGCCAGC CTGGGAGGAC CTTCTAGTCC TAAGAAGAAG AGAAAGGTGG 
2701 CGGCCGCATT AGCCCGAAGA TCTTCGGGCT GATCTCCCAT GTCTCTACTG GTGGTGGTGC 
2761 TTCTTTGGAA TTATTGGAAG GTAAGGAATT GCCAGGTGTT GCTTTCTTAT CCGAAAAGAA 
2821 ATAAATTGAA TTGAATTGAA ATCGATAGAT CAATTTTTTT CTTTTCTCTT TCCCCATCCT 
2881 TTACGCTAAA ATAATAGTTT ATTTTATTTT TTGAATATTT TTTATTTATA TACGTATATA 
2941 TAGACTATTA TTTATCTTTT AATGATTATT AAGATTTTTA TTAAAAAAAA ATTCGCTCCT 
3001 CTTTTAATGC CTTTATGCAG TTTTTTTTTC CCATTCGATA TTTCTATGTT CGGGTTCAGC 
3061 GTATTTTAAG TTTAATAACT CGAAAATTCT GCGTTCGTTA AAGCTAGGCC TCGGATCTTC 
3121 CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGTTT GCGTATTGGG CGCTCTTCCG 
3181 CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC 
3241 ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT 
3301 GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC 
3361 ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA 
3421 ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC 
3481 CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG 
3541 CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC 
3601 TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC 
3661 GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA 
3721 GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT 
3781 ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG 
3841 GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT 
3901 TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT 
3961 TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA 
4021 GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA 
4081 TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC 
4141 CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA 
4201 TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC 
4261 CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA 
4321 GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA 
4381 GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG 
4441 TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC 
4501 GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG 
4561 TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT 
4621 CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT 
4681 CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA 
4741 ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC 
4801 GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC 
4861 CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA 
4921 GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT 
4981 TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT 
5041 TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC 
5101 CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA 
5161 CGAGGCCCTT TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC 
5221 TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG 
5281 GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA TCAGAGCAGA 
5341 TTGTACTGAG AGTGCACCAT AACGCATTTA AGCATAAACA CGCACTATGC CGTTCTTCTC 
5401 ATGTATATAT ATATACAGGC AACACGCAGA TATAGGTGCG ACGTGAACAG TGAGCTGTAT 
5461 GTGCGCAGCT CGCGTTGCAT TTTCGGAAGC GCTCGTTTTC GGAAACGCTT TGAAGTTCCT 
5521 ATTCCGAAGT TCCTATTCTC TAGCTAGAAA GTATAGGAAC TTCAGAGCGC TTTTGAAAAC 
5581 CAAAAGCGCT CTGAAGACGC ACTTTCAAAA AACCAAAAAC GCACCGGACT GTAACGAGCT 
5641 ACTAAAATAT TGCGAATACC GCTTCCACAA ACATTGCTCA AAAGTATCTC TTTGCTATAT 
5701 ATCTCTGTGC TATATCCCTA TATAACCTAC CCATCCACCT TTCGCTCCTT GAACTTGCAT 
5761 CTAAACTCGA CCTCTACATT TTTTATGTTT ATCTCTAGTA TTACTCTTTA GACAAAAAAA 
5821 TTGTAGTAAG AACTATTCAT AGAGTGAATC GAAAACAATA CGAAAATGTA AACATTTCCT 
5881 ATACGTAGTA TATAGAGACA AAATAGAAGA AACCGTTCAT AATTTTCTGA CCAATGAAGA 
5941 ATCATCAACG CTATCACTTT CTGTTCACAA AGTATGCGCA ATCCACATCG GTATAGAATA 
6001 TAATCGGGGA TGCCTTTATC TTGAAAAAAT GCACCCGCAG CTTCGCTAGT AATCAGTAAA 
6061 CGCGGGAAGT GGAGTCAGGC TTTTTTTATG GAAGAGAAAA TAGACACCAA AGTAGCCTTC 
6121 TTCTAACCTT AACGGACCTA CAGTGCAAAA AGTTATCAAG AGACTGCATT ATAGAGCGCA 
6181 CAAAGGAGAA AAAAAGTAAT CTAAGATGCT TTGTTAGAAA AATAGCGCTC TCGGGATGCA 
6241 TTTTTGTAGA ACAAAAAAGA AGTATAGATT CTTTGTTGGT AAAATAGCGC TCTCGCGTTG 
6301 CATTTCTGTT CTGTAAAAAT GCAGCTCAGA TTCTTTGTTT GAAAAATTAG CGCTCTCGCG 
6361 TTGCATTTTT GTTTTACAAA AATGAAGCAC AGATTCTTCG TTGGTAAAAT AGCGCTTTCG 
6421 CGTTGCATTT CTGTTCTGTA AAAATGCAGC TCAGATTCTT TGTTTGAAAA ATTAGCGCTC 
6481 TCGCGTTGCA TTTTTGTTCT ACAAAATGAA GCACAGATGC TTCGTT