Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression.
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 alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-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.
Arís
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Institut de Biologia Fonamental and Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Feliu
J X
JX
Knight
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Coutelle
C
C
Villaverde
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D000595:Amino Acid Sequence; D000818:Animals; D005537:Aphthovirus / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics; D016503:Drug Delivery Systems; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007763:Lac Operon; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D019030:Receptors, Vitronectin / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D001616:beta-Galactosidase