[Trans-splicing of Cys mutated coagulation factor VIII].
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.
Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
Fu-Xiang
FX
Zhu
Ze-Long
ZL
Liu
Jing
J
Miao
Hui-Ge
HG
Qu
Xiao-Yan
XY
Chi
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D019154:Protein Splicing; D014162:Transfection
C451034:B-domain-deleted factor VIII; D004220:Disulfides; D010446:Peptide Fragments; D005169:Factor VIII; D003545:Cysteine