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| Study of the Effects of A and C-Domain Glycosylation Sites on the Secretion and Activity of Recombinant Factor Ⅷ |
| GAO Guang-wei, LI Gui-lin, HUANG Jia-yu, LI Da-wei |
| School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China |
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Abstract Because of the safety concerns of blood sources and whole blood products, genetically engineered recombinant factor Ⅷ have begun to replace blood-derived factor Ⅷ for the treatment of hemophilia and related diseases. However, the relatively low expression level of recombinant factor Ⅷ remains a major technology bottleneck to reduce the cost of recombinant factor Ⅷ production. To explore the possible means to increase recombinant factor Ⅷ expression at molecular level, site-directed mutagenesis techniques were used to generate potential glycosylation sites around A and C domains of a B-domain deleted recombinant F Ⅷ and tested the activity, expression and secretion of various glycosylation constructs. The results showed that generating glycosylation sites at the outer edges of domain A increased secretion of factor Ⅷ while those glycosylation sites generated at the Bip-binding core not only greatly reduced the secretion but also abolished the activity of Factor Ⅷ. Different from A-domain, the glycosylation sites generated in C-domain abolished most factor Ⅷ activity without increasing its secretion regardless whether they were located at the core protein-C binding site or at the edge of C-domain. Taken together, the results suggest that the only glycosylation sites with the potential of increasing factor Ⅷ secretion are located around the edge of A-domain.
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Received: 18 March 2014
Published: 25 October 2014
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