[Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].
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.
Zhu
Fuxiang
F
Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
Liu
Zelong
Z
Qu
Huige
H
Xin
Xiaolin
X
Dong
Hongxin
H
Liu
Xiangqin
X
D053488:DnaB Helicases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D047668:Inteins / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019154:Protein Splicing / Q000502:physiology
C451034:B-domain-deleted factor VIII; D010446:Peptide Fragments; D005169:Factor VIII; D053488:DnaB Helicases