Trans-splicing of Cys mutated coagulation factor VIII.
- Author:
Fu-Xiang ZHU
1
;
Ze-Long LIU
;
Jing MIAO
;
Hui-Ge QU
;
Xiao-Yan CHI
Author Information
1. Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
- Publication Type:Journal Article
- MeSH:
Animals;
COS Cells;
Cercopithecus aethiops;
Cysteine;
genetics;
metabolism;
Disulfides;
metabolism;
Factor VIII;
genetics;
metabolism;
Gene Transfer Techniques;
Genetic Vectors;
Mutation;
Peptide Fragments;
genetics;
metabolism;
Protein Splicing;
Transfection
- From:
Acta Pharmaceutica Sinica
2012;47(6):734-738
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
