Adenovirus type 7 penton -: Purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system

被引:7
作者
Bal, HP
Chroboczek, J
Schoehn, G
Ruigrok, RWH
Dewhurst, S
机构
[1] Univ Rochester, Med Ctr, Dept Microbiol & Immunol, Rochester, NY 14642 USA
[2] Inst Biol Struct, Grenoble, France
[3] European Mol Biol Lab, Grenoble Outstn, Grenoble, France
[4] Univ Rochester, Ctr Canc, Rochester, NY 14627 USA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 19期
关键词
adenovirus; penton base; expression; integrin; gene delivery;
D O I
10.1046/j.1432-1327.2000.01684.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:6074 / 6081
页数:8
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