Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII / 药学学报
Acta Pharmaceutica Sinica
;
(12): 1361-1366, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-353353
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Peptide Fragments
/
Glycosylation
/
Factor VIII
/
Transfection
/
Protein Splicing
/
Bodily Secretions
/
Trans-Splicing
/
Inteins
/
HEK293 Cells
/
Genetics
Limits:
Humans
Language:
Chinese
Journal:
Acta Pharmaceutica Sinica
Year:
2010
Type:
Article
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