title: [Glycosylation modifications improve secretion and activity of intein spliced coagulation factor VIII].
abstract: OBJECTIVE
  to investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FVIII (BDD-FVIII).
  
  
  METHODS
  a total of 226 amino acid residues of FVIII B domain with six potential asparagines-linked glycosylation sites (N6) were incorporated into heavy chain of BDD-FVIII. By dual-vector co-transfer of heavy and light chain genes with fused intein (N6HCIntN and IntCLC) into cultured 293 cells, the amounts of spliced BDD-FVIII protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively.
  
  
  RESULTS
  the amounts of spliced BDD-FVIII protein and activity were up to (123 ± 18) ng/ml and (0.94 ± 0.11) U/ml in supernatant from cells co-transfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy (HCIntN) and light chain (IntCLC) genes [(86 ± 12) ng/ml and (0.65 ± 0.07) U/ml, both P < 0.05]. Spliced BDD-FVIII protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [(18 ± 6) ng/ml and (0.15 ± 0.05) U/ml].
  
  
  CONCLUSIONS
  it demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FVIII and the protein splicing can occur independent of cellular mechanism.
authors.affiliation: Biomedical Institute, 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*; D005822:Genetic Vectors; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D047668:Inteins / Q000235:genetics; D019154:Protein Splicing; D014162:Transfection
chemical_list: D005169:Factor VIII