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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
6
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pubmed:dateCreated |
2002-6-3
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pubmed:abstractText |
We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/3-phosphoinositide-dependent...,
http://linkedlifedata.com/resource/pubmed/chemical/BAD protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Cephalosporins,
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Immunophilins,
http://linkedlifedata.com/resource/pubmed/chemical/Leucine,
http://linkedlifedata.com/resource/pubmed/chemical/MTOR protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Peptide Fragments,
http://linkedlifedata.com/resource/pubmed/chemical/Peptide Library,
http://linkedlifedata.com/resource/pubmed/chemical/Phosphotransferases (Alcohol Group...,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Kinases,
http://linkedlifedata.com/resource/pubmed/chemical/Protein-Serine-Threonine Kinases,
http://linkedlifedata.com/resource/pubmed/chemical/Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Proto-Oncogene Proteins c-bcl-2,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Fusion Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/SMAD3 protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Saccharomyces cerevisiae Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Smad3 Protein,
http://linkedlifedata.com/resource/pubmed/chemical/TOR Serine-Threonine Kinases,
http://linkedlifedata.com/resource/pubmed/chemical/Trans-Activators,
http://linkedlifedata.com/resource/pubmed/chemical/bcl-Associated Death Protein,
http://linkedlifedata.com/resource/pubmed/chemical/beta-Lactamases,
http://linkedlifedata.com/resource/pubmed/chemical/beta-lactamase TEM-1,
http://linkedlifedata.com/resource/pubmed/chemical/nitrocefin
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pubmed:status |
MEDLINE
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pubmed:month |
Jun
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pubmed:issn |
1087-0156
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
20
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
619-22
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pubmed:dateRevised |
2010-11-18
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pubmed:meshHeading |
pubmed-meshheading:12042868-Animals,
pubmed-meshheading:12042868-Carrier Proteins,
pubmed-meshheading:12042868-Cattle,
pubmed-meshheading:12042868-Cell Line,
pubmed-meshheading:12042868-Cephalosporins,
pubmed-meshheading:12042868-DNA-Binding Proteins,
pubmed-meshheading:12042868-Humans,
pubmed-meshheading:12042868-Immunophilins,
pubmed-meshheading:12042868-Kidney,
pubmed-meshheading:12042868-Leucine,
pubmed-meshheading:12042868-Microscopy, Fluorescence,
pubmed-meshheading:12042868-Peptide Fragments,
pubmed-meshheading:12042868-Peptide Library,
pubmed-meshheading:12042868-Phosphotransferases (Alcohol Group Acceptor),
pubmed-meshheading:12042868-Protein Conformation,
pubmed-meshheading:12042868-Protein Folding,
pubmed-meshheading:12042868-Protein Interaction Mapping,
pubmed-meshheading:12042868-Protein Kinases,
pubmed-meshheading:12042868-Protein-Serine-Threonine Kinases,
pubmed-meshheading:12042868-Proteins,
pubmed-meshheading:12042868-Proto-Oncogene Proteins c-bcl-2,
pubmed-meshheading:12042868-Recombinant Fusion Proteins,
pubmed-meshheading:12042868-Reproducibility of Results,
pubmed-meshheading:12042868-Saccharomyces cerevisiae Proteins,
pubmed-meshheading:12042868-Sensitivity and Specificity,
pubmed-meshheading:12042868-Smad3 Protein,
pubmed-meshheading:12042868-TOR Serine-Threonine Kinases,
pubmed-meshheading:12042868-Trans-Activators,
pubmed-meshheading:12042868-bcl-Associated Death Protein,
pubmed-meshheading:12042868-beta-Lactamases
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pubmed:year |
2002
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pubmed:articleTitle |
Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions.
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pubmed:affiliation |
Département de Biochimie, C.P. 6128, Succursale Centre-Ville, Montréal, QC, H3C 3J7, Canada.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't,
Validation Studies
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