High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals

Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, β and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a...

Full description

Saved in:
Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2016-02, Vol.159 (2), p.261-270
Main Authors: Hirashima, Masaki, Imamura, Takayuki, Yano, Kentaro, Kawamura, Ryoichi, Meta, Akihiro, Tokieda, Yoshiyuki, Nakashima, Toshihiro
Format: Article
Language:English
Subjects:
Animals
Biological Products - isolation & purification
Biological Products - metabolism
Blotting, Western
CHO Cells
Cricetinae
Cricetulus
Electrophoresis, Polyacrylamide Gel
Fibrin - metabolism
Fibrinogen - biosynthesis
Fibrinogen - isolation & purification
Humans
Microscopy, Electron, Scanning
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Regular Papers
Thrombin - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, β and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, β and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvv099