Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII.
- Author:
Sang Hwan OH
1
;
Mi Young LEE
;
Dong Weon SONG
Author Information
1. Department of Biochemistry and Molecular Biology, College of Medicine, Yonsei University, Seoul, Korea. shoh@yumc.yonsei.ac.kr
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
recombinant FVIII;
baculovirus;
expression;
mutation
- MeSH:
Animal;
Baculoviridae/genetics;
Blotting, Western;
Cell Line;
Factor VIII/metabolism*;
Factor VIII/genetics;
Factor VIII/chemistry;
Factor VIII/biosynthesis;
Genetic Vectors;
Human;
Mutagenesis, Site-Directed;
Recombinant Proteins/metabolism;
Recombinant Proteins/genetics;
Recombinant Proteins/chemistry;
Recombinant Proteins/biosynthesis;
Spodoptera
- From:Experimental & Molecular Medicine
1999;31(2):95-100
- CountryRepublic of Korea
- Language:English
-
Abstract:
FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.
