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J Biol Chem. 1996 May 3;271(18):10560-8.

Target cell-specific DNA transfer mediated by a chimeric multidomain protein. Novel non-viral gene delivery system.

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1
Institute for Experimental Cancer Research, Tumor Biology Center, Freiburg, Federal Republic of Germany.

Abstract

Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains: 1) an ErbB-2 -specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enable sequence-specific high affinity binding to DNA. Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro. Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner. Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex. Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer.

PMID:
8631856
[Indexed for MEDLINE]
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