High-level expression of functional recombinant human coagulation factor VII in insect cells

Masroori, Nasser; Halabian, Raheleh; Mohammadipour, Mahshid; Roushandeh, Amaneh Mohammadi; Rouhbakhsh, Mahdi; Jahanian-Najafabadi, Ali; Fathabad, Mahdi Edalati; Salimi, Mohamad; Shokrgozar, Mohammad Ali; Roudkenar, Mehryar Habibi

doi:10.1007/s10529-010-0227-7

Cited by 10 publications

(8 citation statements)

References 19 publications

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“…We intend to analyze the expressed proteins by matrixassisted laser desorption ionization-time-of-flight spectroscopy to determine the levels of gammacarboxylation. Bioactive recombinant FVII was expressed in HEK293 cells-which endogenously express human GGCX-by co-transfecting rat Vkorc1 (Wajih et al 2008); it was also expressed in Sf9 cells (another insect cell line) by co-transfecting human GGCX (Masroori et al 2010). Mammalian cells are used to express bioactive vit.K-dependent proteins as therapeutic compounds, including recombinant FVII (Novo Nordisk, Princeton, NJ, USA), recombinant FIX (CSL Behring, King of Prussia, PA, USA), and recombinant activated protein C (Eli Lilly and Company, Indianapolis, IN, USA).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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“…The optimal combination of culture and feed media resulted in 20 mg/L of rFVII expression in CHO cells, a result that is higher than previously reported studies. 2,4,5,23 Moreover, it has been reported that the cell cycles phase, specifically the percentage of cells in the G0/G1 phase, significantly influences the specific productivity and production of recombinant proteins in CHO cells. [16][17][18][19] Many researchers have focused on screening for chemicals that pause the cells in the G0/ G1 phase to enhance protein specific productivity in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

“…Currently, several recombinant protein expression systems, such as Chinese hamster ovary (CHO), HEK 293 and insects cells, have been developed to express rFVII. [2][3][4][5] Moreover, Hwang et al reported using transgenic fish as bioreactors to express rFVII. 6 However, the main problem with rFVII production is its low expression level during culture.…”

Section: Introductionmentioning

confidence: 99%

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

Lin

et al. 2016

Bioengineered

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(2016) Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium, Bioengineered, 7:3, 189-197, DOI: 10.1080/21655979.2016 ABSTRACTSignal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4CF4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

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“…Initially, BHK cells and subsequently the variety of recombinant protein expression systems such as insect and other mammalian cells have been developed as a resource of FVII gene expression [4, 10, 11]. However, in this study, leishmania expression system was used as a host cell.…”

Section: Discussionmentioning

confidence: 99%

Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

Mirzaahmadi

Asaadi-Tehrani

Bandehpour

et al. 2011

BioMed Research International

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The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

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High-level expression of functional recombinant human coagulation factor VII in insect cells

Cited by 10 publications

References 19 publications

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

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

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

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