Rm. Camire et al., Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide, BIOCHEM, 39(46), 2000, pp. 14322-14329
Factor Xa is the serine protease component of prothrombinase, the enzymatic
complex responsible for thrombin generation. Production of recombinant fac
tor X/Xa has proven to be difficult because of inefficient gamma -carboxyla
tion, a critical post-translational modification. The affinities of the vit
amin K-dependent propeptides for the gamma -carboxylase vary over 2 logs, w
ith the propeptide of factor X having the highest affinity followed by the
propeptides of factor VII, protein S, factor LX, protein C, and prothrombin
[Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944], On the basis of t
his observation, it was hypothesized that exchanging the propeptide of fact
or X with one that binds the gamma -carboxylase with a reduced affinity wou
ld enhance gamma -carboxylation by allowing greater substrate turnover, A c
himeric cDNA consisting of the human prothrombin signal sequence and propep
tide followed by mature human factor:X was generated and stably transfected
into HEK 293 cells, and modified factor X was purified from conditioned me
dium, The results indicate that on average 85% of the total factor X produc
ed with the prothrombin propeptide was fully gamma -carboxylated, represent
ing a substantial improvement over a system that employs the native factor
X propeptide, with which on average only 32% of the protein is fully gamma
-carboxylated. These results indicate that the affinity of the gamma -carbo
xylase for the propeptide greatly influences the extent of gamma -carboxyla
tion. It was also observed that regardless of which propeptide sequence is
directing gamma -carboxylation (factor X or prothrombin), two pools of fact
or X are secreted; one is uncarboxylated and a second is fully gamma -carbo
xylated, supporting the notion that the gamma -carboxylase is a processive
enzyme.