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Related Experiment Videos

Expression, purification, and characterization of recombinant human factor X

A E Rudolph1, M P Mullane, R Porche-Sorbet

  • 1Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110-8118, USA.

Protein Expression and Purification
|August 1, 1997
PubMed
Summary

This study developed a novel system for producing recombinant factor X using human kidney cells, yielding a product highly similar to plasma factor X. This advancement facilitates research into factor X structure-function relationships.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Hematology

Background:

  • Factor X is a crucial protein in the blood coagulation cascade.
  • Existing methods for producing recombinant factor X have limitations.
  • Understanding factor X function is vital for treating bleeding disorders.

Purpose of the Study:

  • To develop an optimized system for producing functional recombinant factor X.
  • To compare the properties of recombinant factor X with human plasma factor X.
  • To establish a reliable method for recombinant factor X production for further research.

Main Methods:

  • Optimization of expression systems, comparing human kidney (293) cells and baby hamster kidney (BHK) cells.
  • Genetic modification of factor X for enhanced expression in 293 cells.

Related Experiment Videos

  • Purification using a calcium-dependent monoclonal antibody against the gla domain.
  • Functional characterization through activation assays and inhibition studies.

    • Main Results:

    • Human 293 cells are superior to BHK cells for functional factor X expression.
    • A specific residue substitution (Thr-->Arg) is required for propeptide removal.
    • Recombinant factor X exhibits comparable activation kinetics and enzymatic activity to plasma factor X.
    • Inhibition profiles by antithrombin and tissue factor pathway inhibitor are equivalent for both forms.

    Conclusions:

    • A robust system for producing high-quality recombinant factor X has been established.
    • The recombinant factor X produced is functionally equivalent to plasma-derived factor X.
    • This recombinant factor X is suitable for detailed structure-function studies using mutational analysis.