Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method for coupling proteins to plasmid expression vectors is presented. Biotin was covalently attached to a plasmid expression vector containing a chloramphenicol acetyltransferase (CAT) gene. The specific label was one biotin per 100 bp. An electrophoretic mobility shift assay showed that the plasmid was capable of binding multiple streptavidin molecules. When transfected into mouse fibroblasts, the biotinylated plasmid retained 40% of the native plasmid's biological activity, as determined by CAT assay, and was not affected by the binding of streptavidin. The method allows for attachment of any protein to plasmid DNA expression vector while retaining biological function. Hybrid plasmids in which the transcription cassettes were kept free of biotin label were constructed by digesting biotinylated and unbiotinylated plasmids at sites outside the transcription cassette and re-ligating the digestion products. Electron microscopy studies show that the ligation products formed large tangled assemblages of plasmid DNA. When equimolar (with respect to gene number) amounts of these large hybrid biotinylated plasmids were transfected into mouse fibroblasts by means of calcium phosphate precipitation, an increase in CAT expression 25-fold greater than that of original biotinylated plasmid was observed. Slot-blot analysis of total DNA extracted from transfected cells shows that this enhanced activity was not due to increased transfection efficiency. Receptor-mediated delivery could not be shown when a complex comprising biotinylated asialoglycoprotein/streptavidin/biotinylated CAT expression vector was placed in media containing Hep G2 cells.
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PMID:Novel biotinylated plasmid expression vectors retain biological function and can bind streptavidin. 888 15

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.
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PMID:Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. 1545 38