Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli
Identifieur interne : 001219 ( Main/Exploration ); précédent : 001218; suivant : 001220Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli
Auteurs : Weiyu Wang [République populaire de Chine] ; Jian Luo [République populaire de Chine] ; Lining Xu [République populaire de Chine] ; Jianping Zeng [République populaire de Chine] ; Limin Cao [République populaire de Chine] ; Jiahong Dong [République populaire de Chine] ; Shouwang Cai [République populaire de Chine]Source :
- Cell Biochemistry and Function [ 0263-6484 ] ; 2013-12.
Abstract
Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni2+ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni2+ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/cbf.2958
Affiliations:
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In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni2+ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni2+ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country><region><li>Hubei</li></region><settlement><li>Pékin</li><li>Wuhan</li></settlement></list><tree><country name="République populaire de Chine"><noRegion><name sortKey="Wang, Weiyu" sort="Wang, Weiyu" uniqKey="Wang W" first="Weiyu" last="Wang">Weiyu Wang</name></noRegion><name sortKey="Cai, Shouwang" sort="Cai, Shouwang" uniqKey="Cai S" first="Shouwang" last="Cai">Shouwang Cai</name><name sortKey="Cao, Limin" sort="Cao, Limin" uniqKey="Cao L" first="Limin" last="Cao">Limin Cao</name><name sortKey="Dong, Jiahong" sort="Dong, Jiahong" uniqKey="Dong J" first="Jiahong" last="Dong">Jiahong Dong</name><name sortKey="Luo, Jian" sort="Luo, Jian" uniqKey="Luo J" first="Jian" last="Luo">Jian Luo</name><name sortKey="Wang, Weiyu" sort="Wang, Weiyu" uniqKey="Wang W" first="Weiyu" last="Wang">Weiyu Wang</name><name sortKey="Xu, Lining" sort="Xu, Lining" uniqKey="Xu L" first="Lining" last="Xu">Lining Xu</name><name sortKey="Zeng, Jianping" sort="Zeng, Jianping" uniqKey="Zeng J" first="Jianping" last="Zeng">Jianping Zeng</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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