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[Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII.] [Ariticle in Chinese]

ZHU Fu-Xiang^*, LIU Ze-Long, QU Hui-Ge, CHI Xiao-Yan

Life Science College of Ludong University, Yantai 264025, China

Abstract

Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIIIgene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediatedprotein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally splicedfVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcinefVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. Apair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfectedinto COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatantof cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of splicedhfVIII secreted from the cells co-transfected with human fVIII [(184±34 ng/mL) vs (48±12) ng/mL, P<0.01; (1.18±0.22) IU/mL vs(0.31±0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of inteinsplicedHP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separatelytransfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independentof cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularlyspliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. Theseresults provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIIIgene in animal models.

Key words: human/porcine hybrid factor VIII; secretion; intein; protein trans-splicing

Received: 2010-04-12　　Accepted: 2010-05-26

Corresponding author: 朱甫祥　　E-mail: fuxiangmail@163.com

Citing This Article：

ZHU Fu-Xiang, LIU Ze-Long, QU Hui-Ge, CHI Xiao-Yan. [Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (4): 373-381 (in Chinese with English abstract).