title: Expression of scFv-Mel-Gal4 triple fusion protein as a targeted DNA-carrier in Escherichia Coli.
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 Ni(2+) 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 Ni(2+) 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.
authors.affiliation: Hospital & Institute of Hepatobiliary Surgery, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853, China; Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors.forename: Weiyu
authors.initials: W
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Luo
authors.forename: Lining
authors.initials: L
authors.lastname: Xu
authors.forename: Jianping
authors.initials: J
authors.lastname: Zeng
authors.forename: Limin
authors.initials: L
authors.lastname: Cao
authors.forename: Jiahong
authors.initials: J
authors.lastname: Dong
authors.forename: Shouwang
authors.initials: S
authors.lastname: Cai
mesh_terms: D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004337:Drug Carriers; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D037263:Asialoglycoprotein Receptor; D004268:DNA-Binding Proteins; D004337:Drug Carriers; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D014157:Transcription Factors; D004247:DNA
keywords: DNA; Gal4 protein; Melittin; gene transfer; hepatocyte-specific delivery; recombinant protein; sequence-specific binding; single-chain Fv