In vitro carboxylation of a blood coagulation factor IX precursor produced by recombinant-DNA technology.
Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.
Soute
B A
BA
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Balland
A
A
Faure
T
T
de la Salle
H
H
Vermeer
C
C
D000818:Animals; D019735:Carbon-Carbon Ligases; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000302:isolation & purification / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D005164:Factor IX; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase