Characterization of purified recombinant fibrinogen: partial phosphorylation of fibrinopeptide A

Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months...

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Bibliographic Details
Published in:Biochemistry (Easton) 1993-01, Vol.32 (1), p.107-113
Main Authors: Binnie, Cameron G, Hettasch, Joann M, Strickland, Elizabeth, Lord, Susan T
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Base Sequence
Biological and medical sciences
Blotting, Western
CHO Cells - metabolism
Cloning, Molecular
Cricetinae
DNA - genetics
Enzyme-Linked Immunosorbent Assay
Fibrinogen - genetics
Fibrinogen - isolation & purification
Fibrinogen - metabolism
Fibrinolysin - metabolism
Fibrinopeptide A - metabolism
Fundamental and applied biological sciences. Psychology
Glycoproteins
Humans
Molecular Sequence Data
Phosphorylation
Proteins
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Thrombin - metabolism
Transfection
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Summary:Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00052a015