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

Abstract

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.

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

“…23 Low expression of rFVII in these studies may be caused by the low cell density and short culture time of adhered cells. Therefore, suspension culture already the main method used to produce therapeutic proteins should be developed and applied to rFVII expression.…”

“…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.…”

Genetic polymorphisms in the FVII gene is associated with lower extremity deep venous thrombosis: A case‐control study

“…FVII is consisted of 406 residues of a single peptide chain, with a molecular weight of 50 KD, and the plasma levels of FVII are influenced by both genetic and environmental factors . FVII is one of the key molecules involved in blood clot formation, which plays an important role in blood coagulation process and induces the generation of fibrin during the coagulation process . Common polymorphisms (−323 0/10 bp, −401 G/T, −402 G/A) at the FVII gene locus may modulate FVII gene transcription, thus exerting an effect on plasma FVII protein levels .…”

Clonagem e expressão do fator VII de coagulação sanguínea em linhagens celulares humanas

“…Além disso, os trabalhos publicados até o momento também possuem em comum o fato de utilizarem vetores não virais e a utilização de drogas como pressão seletiva para geração de uma linhagem celular mais estável (WAHJI et al, 2008;XIAO et al, 2013). Em relação à produção, a maioria dos trabalhos mostrou uma expressão em torno de 270 a 500 ng/mL de FVIIr, com exceção do trabalho de Kemball-Cook (1994), que relata a produção de 5000 ng/mL (KEMBALL-COOK et al,…”