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Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide
R M Camire1, P J Larson, D W Stafford
1Department of Pediatrics and Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Biochemistry
|November 23, 2000
Summary
Enhancing recombinant factor X production involves modifying its propeptide. Replacing factor X
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Recombinant factor X/Xa production is hindered by inefficient gamma-carboxylation.
- Gamma-carboxylation is a critical post-translational modification for factor Xa function.
- Propeptide affinity for gamma-carboxylase varies significantly, impacting efficiency.
Purpose of the Study:
- To enhance gamma-carboxylation of recombinant factor X.
- To investigate the role of propeptide-gamma-carboxylase affinity in carboxylation efficiency.
- To improve the production of functional recombinant factor Xa.
Main Methods:
- Generated a chimeric cDNA with human prothrombin signal sequence and propeptide fused to mature human factor X.
- Stably transfected the chimeric cDNA into HEK 293 cells.
- Purified modified factor X from conditioned medium.
Main Results:
- Factor X produced with the prothrombin propeptide showed 85% full gamma-carboxylation.
- Native factor X propeptide resulted in only 32% full gamma-carboxylation.
- Observed two pools of secreted factor X: uncarboxylated and fully gamma-carboxylated.
Conclusions:
- Propeptide affinity for gamma-carboxylase directly influences the extent of gamma-carboxylation.
- Modifying propeptide sequence can significantly improve recombinant protein production.
- Gamma-carboxylase likely acts as a processive enzyme.