title: Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.
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.
authors.affiliation: Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, Sichuan, China.
authors.forename: W
authors.initials: W
authors.lastname: Xiao
authors.forename: C Q
authors.initials: CQ
authors.lastname: Li
authors.forename: X P
authors.initials: XP
authors.lastname: Xiao
authors.forename: F Z
authors.initials: FZ
authors.lastname: Lin
mesh_terms: D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000235:genetics / Q000502:physiology; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005167:Factor VII / Q000096:biosynthesis / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; D005167:Factor VII