Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system.
Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino-acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C-terminal fusion with glutathione S-transferase (GST-Ad7PB) and was purified by single-step affinity chromatography. Both GST-Ad7PB and cleaved (GST-free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST-Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C-terminus of this linker peptide. GST-Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD-containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin-mediated uptake of the GST-Ad7PB-FK20-DNA complexes. We propose that Ad7 pentons can be developed into integrin-specific gene delivery agents.
Bal
H P
HP
Department of Microbiology and Immunology, University of Rochester Medical Center, NY 14642, USA.
Chroboczek
J
J
Schoehn
G
G
Ruigrok
R W
RW
Dewhurst
S
S
D000260:Adenoviruses, Human / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D036022:Capsid Proteins; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism; D002846:Chromatography, Affinity; D004847:Epithelial Cells / Q000378:metabolism / Q000821:virology; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D016023:Integrins / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011991:Receptors, Virus / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D036022:Capsid Proteins; D018834:Chaperonin 60; D016023:Integrins; D011991:Receptors, Virus; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C064660:integrin alphaVbeta5; C043322:penton protein, adenovirus; D005982:Glutathione Transferase; D015951:Factor Xa