Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain

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Author:
ZHU Fu-Xiang(Life Science College of Ludong University, Yantai 264025, China)
LIU Ze-Long(Life Science College of Ludong University, Yantai 264025, China)
QU Hui-Ge(Life Science College of Ludong University, Yantai 264025, China)
CHI Xiao-Yan(Life Science College of Ludong University, Yantai 264025, China)
Journal Title:
ACTA PHYSIOLOGICA SINICA
Issue:
Volume 61, Issue 06, 2009
DOI:
Key Word:
coagulation factor VIII; secretion; intein; protein trans-splicing

Abstract: Coagulation factor Ⅷ (fⅧ) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fⅧ is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fⅧ gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe~(309)→Ser mutant full-length fⅧ (F309SfⅧ) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfⅧ is proven to be beneficial to its secretion. The F309SfⅧ gene was broken into heavy and light chains before Ser~(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfⅧ protein band by Western blot with a polyclonal antibody against fⅧ. The amounts of secreted F309SfⅧ protein in culture supernatants and their bioactivities were (71 ±9) ng/mL and (0.38±0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfⅧ antigen and fⅧ activity [(25±6) ng/mL and (0.12±05) IU/ mL], indicating the F309SfⅧ could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135±10) ng/mL vs (37±7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfⅧ gene with improved secretion of fⅧ providing an alternative approach to circumvent the packaging limitation of AAV for F309SfⅧ gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.

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