Abstract：
:The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Hays LB,Chen YS,Hu JCdoi
10.2144/00292st04subject
Has Abstractpub_date
2000-08-01 00:00:00pages
288-90, 292, 294 passimissue
2eissn
0736-6205issn
1940-9818journal_volume
29pub_type
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