The Gene Expression of Coagulation Factor VIII in Mammalian Cell Lines

Chen, Chun; Fang, Xiangdong; Zhu, Jiang; Wu, Xiang-Fu; Zhang, Zu-chuan; Gu, Jianxin; Wang, Zhenyi; Chi, Cheng-Wu

doi:10.1016/s0049-3848(99)00031-6

Cited by 22 publications

(13 citation statements)

References 25 publications

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“…The highest expression level was obtained in the CHO cells. In this work, expression levels were 40 to 50-fold higher (400 ng/mL) than obtained by Haack et al [32] and 2-fold higher than Chen et al [33]. …”

Section: Discussioncontrasting

confidence: 53%

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Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

et al. 2011

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BackgroundHemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C.ResultsWe observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.ConclusionSerum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

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Section: Discussioncontrasting

confidence: 53%

“…The authors presented a kinetic expression profile but the levels reached were low (around 8-10 ng/mL). Chen et al [33] studied the rFVIII transient expression in different cell lines (SMMC-7721, A549, Cos-7 and CHO) and found rFVIII levels in the range of 24 to 201 ng/mL. The highest expression level was obtained in the CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

et al. 2011

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“…The higher expression levels of coagulation factor VIII when coexpressed with VWF are thought to be due to the proteolysis-protective properties of VWF [116]. This property may be important for the protection of Vtg during passage in the systemic circulation, as well as during the differential proteolysis of the Yps of marine teleosts during final oocyte maturation [5,65].…”

Section: Von Willebrand Factormentioning

confidence: 99%

Vertebrate Yolk Complexes and the Functional Implications of Phosvitins and Other Subdomains in Vitellogenins1

Finn

2007

Biology of Reproduction

155

121

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In nonplacental or nontrophotenic vertebrates, early development depends on the maternal provision of egg yolk, which is mainly derived from large multidomain vitellogenin (Vtg) precursors. To reveal the molecular nature of the protein pools in vertebrate oocytes, published data on the N-termini of yolk proteins has been mapped to the deduced primary structures of their parent Vtgs. The available evidence shows that the primary cleavage sites of Vtgs are conserved, whereas the cleavage products exist as multidomain variants in the yolk protein pool. The serine-rich phosvitin (Pv) domains are linearly related to the molecular masses of the lipovitellin heavy chain. The 3-D localization of Pv maps to the outer edges of the Vtg monomer, where it is proposed to form amphipathic structures that loop up over the lipid pocket. At this locus, it is proposed that Pv stabilizes the nascent Vtg while it receives its lipid cargo, thereby facilitating the hepatic loading and locking of lipid within the Vtg (C-sheet)-(A-sheet)-(LvL) cavity, and enhances its solubility following secretion to the circulating plasma. The C-terminal regions of Vtgs are homologous to human von Willebrand factor type D domains (Vwfd), which are conserved cysteine-rich molecules with homologous regions that are prevalent in Vtgs, lipophorins, mucins, integrins, and zonadhesins. Unlike human VWFD, lower vertebrate Vwfds do not contain RGD motifs, which are associated with extracellular matrix binding. Although its function in Vtg is unknown, the lubricant properties associated with mucins and the cell adhesion properties associated with integrins and zonadhesins implicate Vwfd in the genesis of hemostatic platelet aggregation. Similarly, the proteolytic inhibitory properties associated with the binding of factor VIII in humans suggest that Vwfd stabilizes Vtg during passage in the systemic circulation.

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“…[3][4][5][6][7][8][9][10][11][12] As novas técnicas de engenharia celular e biotecnologia avançaram muito nas últimas décadas e entre elas a produção de anticorpos monoclonais utilizados tanto para o aprimoramento diagnóstico em rotinas laboratoriais como na produção de medicamentos e purificação de proteínas utilizadas na terapêutica. [13][14][15][16][17] Os concentrados de FVIII purificados através de anticorpos monoclonais (denominados produtos ultrapuros) colocados no mercado no início dos anos 90 vieram solucionar complicações desastrosas no tratamento da hemofilia, pois se trata de terapêutica eficaz e segura apresentando alta especificidade, segurança viral e redução de efeitos colaterais. Porém estes produtos só podem ser adquiridos no mercado internacional a um custo bastante elevado, pois inexiste no Brasil a produção destes anticorpos voltados a esta finalidade.…”

Section: Introductionunclassified

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

Rossi-Ferreira¹,

Deffune²,

Thomazini-Santos³

et al. 2006

Rev. Bras. Hematol. Hemoter.

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Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm) utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator IntroduçãoA hemofilia é uma doença causada pela deficiência do Fator VIII, uma glicoproteína que participa na via intrínse-ca da coagulação sangüínea e tem como etiologia a herança genética, ligada ao cromossomo X, ocorrendo exclusivamente em homens. A severidade da doença está diretamente relacionada com a extensão da deficiência de FVIII, que é classificada em hemofilia severa, moderada e branda. O tratamento consiste na reposição do Fator VIII através de transfusões sangüíneas.Em 1959, Pool & Robinson, 1 utilizando o método de Cohn 2 na produção de sedimento insolúvel (crioprecipitado) através de processos de congelamento e descongelamento lento, purificaram as proteínas plasmáticas com atividade anti-hemofílica. O crioprecipitado foi utilizado na terapia da hemofilia até a início da década de 80, quando surgiu o dramático problema da contaminação pelo vírus do HIV, detectado no sangue de hemofílicos que receberam crioprecipitado plasmático. A partir daí iniciaram-se inúmeras pesquisas com o objetivo de introduzir novos produtos na terapêutica da hemofilia. [3][4][5][6][7][8][9][10][11][12]

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The Gene Expression of Coagulation Factor VIII in Mammalian Cell Lines

Cited by 22 publications

References 25 publications

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Vertebrate Yolk Complexes and the Functional Implications of Phosvitins and Other Subdomains in Vitellogenins1

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

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