Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
; (12): 1101-1106, 2009.
Article
Dans Zh
| WPRIM
| ID: wpr-296950
Responsable en Bibliothèque :
WPRO
ABSTRACT
We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
Fragments peptidiques
/
Physiologie
/
Facteur VIII
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Chimie
/
Épissage des protéines
/
Intéines
/
Escherichia coli
/
DnaB Helicases
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Génétique
/
Métabolisme
Type d'étude:
Prognostic_studies
langue:
Zh
Texte intégral:
Chinese Journal of Biotechnology
Année:
2009
Type:
Article