title: [Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII].
abstract: We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.
authors.affiliation: Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
authors.forename: Fu-Xiang
authors.initials: FX
authors.lastname: Zhu
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: D005169:Factor VIII / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D047668:Inteins; D009154:Mutation; D010446:Peptide Fragments / 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