Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli.
- Author:
Fuxiang ZHU
1
;
Zelong LIU
;
Huige QU
;
Xiaolin XIN
;
Hongxin DONG
;
Xiangqin LIU
Author Information
1. Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
- Publication Type:Journal Article
- MeSH:
DnaB Helicases;
genetics;
Escherichia coli;
genetics;
metabolism;
Factor VIII;
chemistry;
genetics;
metabolism;
Inteins;
physiology;
Peptide Fragments;
chemistry;
genetics;
metabolism;
Protein Splicing;
physiology
- From:
Chinese Journal of Biotechnology
2009;25(7):1101-1106
- CountryChina
- Language:Chinese
-
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.
