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Exploiting viral cell‐targeting abilities in a single polypeptide, non‐infectious, recombinant vehicle for integrin‐mediated DNA delivery and gene expression
Author(s) -
Arís Anna,
Feliu Jordi X.,
Knight Andrew,
Coutelle Charles,
Villaverde Antonio
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000620)68:6<689::aid-bit13>3.0.co;2-a
Subject(s) - recombinant dna , transfection , microbiology and biotechnology , biology , gene delivery , myc tag , reporter gene , dna , flag tag , plasmid , viral vector , gene , gene expression , fusion protein , biochemistry
A recombinant, multifunctional protein has been designed for optimized, cell‐targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin α V β 3 binding, a DNA‐condensing poly‐L‐lysine domain, and a complete, functional β‐galactosidase protein that serves simultaneously as purification tag and DNA‐shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase‐reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell‐targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid‐based DNA‐condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non‐viral modular DNA transfer vectors that conserve the cell‐target‐ ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 689–696, 2000.