並列タイトル等ショウジョウバエシュナイダーS2細胞における哺乳動物γ-グルタミルカルボキシラーゼの共発現およびvit.Kサイクルの修飾による活性化ヒト凝固第VII因子合成の成功
一般注記doctoral
医学系研究科
Mammalian gamma-glutamyl carboxylase and reduced vitamin K are indispensable for synthesis of mature mammalian vitamin K dependent proteins including some of blood coagulation factors (factors II, VII, IX, and X). It was well known that Drosophila melanogaster expressed gamma-glutamyl carboxylase and possessed a vit.K cycle although native substrates for them have not been identified yet. Despite the potential capability of gamma calboxylation in Drosophila melanogaster derived cells such as S2 cells, Drosophila gamma-glutamyl carboxylase failed to gamma carboxylate a peptide fused to the human coagulation factor IX propeptide. Thus, it had been believed that the Drosophila system was not adequate to synthesize mammalian vit.K dependent proteins. Indeed, we previously attempted to synthesize biologically active factor VII in S2 cells although we were not able to obtain it. However, recently, a successful transient expression of biologically active human factor IX from S2 cells were reported. In the present study, several expression vectors which enables to express mammalian GGCX, VKORC1, and/or PDIA2 along with F7 were developed. S2 cells transfected with pMKA85, pMAK86, and pMAK219 successfully synthesized active FVII. Thus, mammalian GGCX was indispensable to synthesize active FVII while mammalian VKORC1 and PDIA2 were not critical but supportive factors for S2 cells.
DOIinfo:doi/10.1007/s10616-016-0059-y
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