Novel biotinylated plasmid expression vectors retain biological function and can bind streptavidin.
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.
Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06030, USA. PXL24@po.cwru.edu
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Leahy
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Carmichael
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Rossomando
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D051379:Mice; D008854:Microscopy, Electron; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism*; D019809:Streptavidin
D001426:Bacterial Proteins; D001710:Biotin; D019809:Streptavidin