Premium
The carboxylation efficiency of the vitamin K‐dependent clotting factors: studies with factor IX
Author(s) -
BLOSTEIN M.,
CUERQUIS J.,
LANDRY S.,
GALIPEAU J.
Publication year - 2008
Publication title -
haemophilia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.213
H-Index - 92
eISSN - 1365-2516
pISSN - 1351-8216
DOI - 10.1111/j.1365-2516.2008.01828.x
Subject(s) - carboxylation , hek 293 cells , protein precursor , factor ix , clotting factor , in vitro , recombinant dna , haemophilia , transfection , medicine , coagulation , biochemistry , microbiology and biotechnology , gene , chemistry , biology , pediatrics , catalysis
Summary. Haemophilia B is characterized by a deficiency of the γ‐carboxylated protein, factor IX (FIX). As a first step to optimize a gene therapy strategy to treat haemophilia B, we employed a previously described approach ( Biochemistry 2000;39: 14322) of altering the propeptide of vitamin K‐dependent proteins in vitro , to improve the carboxylation efficiency of FIX. Both native FIX and FIX with a prothrombin propeptide (proPT‐FIX) produced recombinant FIX in vitro following transfection of their cDNAs into human embryonic kidney (HEK) 293 cells. Using hydroxyapatite chromatography to separate carboxylated from uncarboxylated FIX, we are able to show that >90% of FIX is γ‐carboxylated and that substituting the propeptide of prothrombin into FIX does not further increase the relative amounts of carboxylated material. These results demonstrate that the nature of the propeptide, per se is not the sole determinant of optimal carboxylation of FIX in our expression system in HEK 293 cells.