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Cloning and recombinant expression of mouse coagulation factor X
H H Heidtmann1, R E Kontermann
1Zentrum für Innere Medizin, Abt. Hämatologie und Onkologie, Philipps-Universität Marburg, Germany.
Abstract:
Engineering of recombinant coagulation factor X variants, which can be activated by tumor-associated proteinases may lead to the development of new therapeutic molecules. However, the evaluation of such variants requires an appropriate animal model. Therefore, we isolated the complete coding sequence of mouse coagulation factor X from mouse liver cDNA by polymerase chain reaction. The deduced amino acid sequence codes for a prepro protein of 481 amino acids homologous to factor X sequences from various species. Recombinant mouse factor X was expressed in human embryonic kidney cells and secreted into cell culture supernatant as zymogen, which could be converted to catalytically active factor Xa by Russell's viper venom. Purified recombinant mouse factor X restored coagulation in human factor X deficient plasma, demonstrating that mouse factor X is able to functionally interact with the human blood coagulation system. Recombinant mouse factor X opens the possibility to analyze therapeutically useful variants in the mouse system.
Insights
Researchers developed recombinant mouse coagulation factor X (FX) for therapeutic applications. This engineered mouse FX functions within the human coagulation system, enabling its study in a relevant animal model.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Developing novel therapeutic molecules targeting coagulation factor X (FX) requires effective animal models.
- Tumor-associated proteinases offer a potential activation pathway for engineered FX variants.
Purpose of the Study:
- To isolate and characterize mouse coagulation factor X (FX) for use in preclinical studies.
- To establish a functional recombinant mouse FX suitable for evaluating therapeutic variants in vivo.
Main Methods:
- Isolation of mouse coagulation factor X (FX) coding sequence via PCR from liver cDNA.
- Expression of recombinant mouse FX in human embryonic kidney cells.
- Activation of recombinant mouse FX using Russell's viper venom and functional assessment in human FX-deficient plasma.
Main Results:
- A 481-amino acid prepro protein sequence homologous to known FX was deduced.
- Recombinant mouse FX was secreted as a zymogen and could be activated to FXa.
- Purified recombinant mouse FX functionally reconstituted coagulation in human FX-deficient plasma.
Conclusions:
- Recombinant mouse coagulation factor X (FX) is functionally active and interacts with the human coagulation system.
- This engineered mouse FX provides a valuable tool for analyzing therapeutic FX variants in a mouse model.
- Enables the development of novel FX-based therapeutics for various medical conditions.