title: [Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system].
abstract: In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
authors.affiliation: College of Life Science, Ludong University, Yantai 264025, China. fuxiangmail@163.com
authors.forename: Fu-Xiang
authors.initials: FX
authors.lastname: Zhu
authors.forename: Shu-De
authors.initials: SD
authors.lastname: Yang
authors.forename: Ze-Long
authors.initials: ZL
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Miao
authors.forename: Hui-Ge
authors.initials: HG
authors.lastname: Qu
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Chi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D047668:Inteins; D016350:Leucine Zippers; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D020040:Trans-Splicing; D014162:Transfection
chemical_list: C451034:B-domain-deleted factor VIII; D010446:Peptide Fragments; D005169:Factor VIII