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Cloning and characterization of a cDNA encoding murine coagulation factor X
Z Liang1, A Cooper, M E DeFord
1Department of Chemistry and Biochemistry and the Center for Transgene Research, University of Notre Dame, Indiana 46556, USA.
Thrombosis and Haemostasis
|July 31, 1998
Summary
Researchers successfully cloned and expressed the gene for mouse coagulation factor X (fX). This mouse fX protein demonstrates clotting activity, similar to human and rat forms, advancing research in hemostasis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Coagulation factor X (fX) is a critical serine protease in the blood coagulation cascade.
- Understanding the genetic and functional characteristics of fX across species is important for comparative biology and potential therapeutic development.
Purpose of the Study:
- To isolate and characterize the complementary DNA (cDNA) encoding murine coagulation factor X (fX).
- To express the mouse fX cDNA and verify its functional activity.
Main Methods:
- cDNA library construction from murine liver mRNA using lambdaZap.
- Sequencing and analysis of the isolated cDNA for open reading frame and protein domains.
- Expression of mouse fX cDNA in human embryonic kidney 293 cells.
- Functional assay of expressed fX using a clotting assay with human fX-deficient plasma.
Main Results:
- Isolated and reconstructed a 1486-base cDNA encoding murine fX.
- The cDNA contains an open reading frame for a 40-amino acid signal/propeptide and a 441-residue mature protein.
- The mature mouse fX protein shows high homology to human and rat fX, with conserved protein domains.
- Expressed mouse fX in HEK 293 cells demonstrated measurable clotting activity.
Conclusions:
- The complete cDNA sequence for murine coagulation factor X has been determined.
- The expressed murine fX is functionally active, confirming its role in hemostasis.
- The high conservation of fX across species suggests a fundamental biological role.