Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells

Abstract

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 carboxylation in D. 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 was reported. In the present study, several expression vectors which enable expressing 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.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Recently, the coexpression of mammalian GGCX, VKORC1, and/or protein disulfide isomerase family A member 2 (PDIA2) enabled the recovery of active rhFVII in S2 cells (Nagahashi, Umemura, Kanayama, & Iwaki, 2017).…”

Produção do fator VII da coagulação sanguínea recombinante em células humanas

“…A expressão e atividade dos fatores dependentes de vitamina K (II, VII, IX, X) são afetadas diretamente por MPTs, tais como a gama-carboxilação do resíduo amino-terminal do domínio Gla, N e O-glicosilações, além da remoção do peptídeo sinal e propetídeo. A gama-carboxilação é responsável pela atividade biológica, que pode ser aumentada por meio de diferentes fatores (VANTANDOOST; PAKDAMAN, 2016), dentre eles a adição de vitamina K (TIE et al 2011;TIE;STAFFORD, 2016;NAGAHASHI et al 2017;WANG et al 1995), cofator envolvido na gama-carboxilação de proteínas dependentes de vitamina K (HAMMED et al 2013). A comparação entre a expressão de FVIIr total pela célula HKB-11 cultivada em meio com suplementação de 1,0 e 2,5 µg/mL, indicou que o aumento na concentração de vitamina K não levou a um aumento na expressão e atividade biológica do FVIIr, nos tempos analisados.…”