Expression and Purification of Recombinant Rabbit Factor VII

Ruiz, Sonia M.; Sridhara, Sampath; Blajchman, Morris A.; Clarke, Bryan J.

doi:10.1016/s0049-3848(99)00227-3

Cited by 16 publications

(23 citation statements)

References 33 publications

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“…The purification of rabbit rFVII was reported by Ruiz et al (2000) who utilized pCMV5 for expressing rabbit rFVII in the human 293 cell line. The rabbit rFVII was purified using a variation of the barium citrate precipitation technique followed by DEAE sepharose FF and benzamidine agarose column chromatography (Gassmann et al, 1990;Ruiz et al, 2000). Commercially available human rFVIIa (NovoSeven) was produced in BHK cells and purified by consecutive chromatography steps, including immunoaffinity chromatography, by using murine monoclonal antibodies (Halabian et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Xiao¹,

Li²,

Xiao³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 μg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 μg/ mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

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Section: Discussionmentioning

confidence: 99%

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Xiao¹,

Li²,

Xiao³

et al. 2013

Genet. Mol. Res.

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“…This generates thrombin that activates platelets and via a cascade process cleaves fibrinogen and factor XIII (Chart 1). Studies that confirm homologies between the coagulation cascade of humans, rabbits, and mice include research by James et al (1997), Marx et al (2000), Ruiz et al (2000), and Humphries et al (Comparative analysis of certain coagulation factors between human and rabbit blood samples, submitted for publication). James et al (1997) showed that intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X, and that mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma.…”

Section: Introductionmentioning

confidence: 99%

Comparative Ultrastructural Analyses of Mouse, Rabbit, and Human Platelets and Fibrin Networks

Pretorius

Humphries

Ekpo

et al. 2007

Microscopy Res & Technique

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Platelets and fibrin play an important role in the coagulation process, where they are involved in the maintenance of hemostasis. Fibrin dysfunction is associated with the development of vascular complications, while proneness to the formation of tight and rigid fibrin networks is independently associated with thrombotic disease. Here we investigate the ultrastructure of human, rabbit, and mouse platelets and fibrin networks, using the scanning electron microscope. Human and rabbit fibrin and platelets, with regards to morphology as well as size of major and minor fibers compare well with each other. However, mouse fibers are much thinner and form a flimsy branching network. Platelet aggregate morphology of all three species compare well with each other. We conclude that rabbit platelet and fibrin networks could be used successfully when studying the effect of pharmaceutical products in preclinical trials, when looking at the effects of these products on morphology and ultrastructure.

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“…Recently, we established a CHO cell line expressing hFVIIa by cotransfection of vectors harboring hFVII and hepsin genes, but the yield was lower than 500 ng/ ml (Halabian et al 2009). However, in all mentioned studies the yield has been lower than 1 lg/ml.week except in Ruiz et al (2000) (5-10 lg/ml.week).…”

Section: Discussionmentioning

confidence: 83%

“…Kemball-Cook et al (1994) expressed rFVII in a CHO cell line. Ruiz et al (2000) reported the expression of rabbit rFVII, utilizing pCMV5 vector for cloning and expression of the rabbit rFVII in human 293T cell line. Commercially available activated recombinant human coagulation factor VII (rFVIIa) or NovoSeven is produced in baby hamster kidney (BHK) cells and purified by consecutive chromatography steps, including immunoaffinity chromatography using murine monoclonal antibodies.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2010

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Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.

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Expression and Purification of Recombinant Rabbit Factor VII

Cited by 16 publications

References 33 publications

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Comparative Ultrastructural Analyses of Mouse, Rabbit, and Human Platelets and Fibrin Networks

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

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