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Related Experiment Videos

Purification of recombinant human tissue factor

L R Paborsky1, K M Tate, R J Harris

  • 1Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.

Biochemistry
|October 3, 1989
PubMed
Summary

Recombinant tissue factor (TF) produced in E. coli is functional, even without glycosylation. A variant with a mutated cytoplasmic cysteine significantly reduces disulfide-linked aggregates, maintaining TF activity.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Tissue Factor (TF) is a critical membrane-bound protein initiating the coagulation cascade.
  • TF acts as a cofactor for serine protease factor VII (fVII).
  • Production of recombinant TF (rTF) is essential for research and therapeutic applications.

Purpose of the Study:

  • To characterize recombinant human TF (rTF) produced in different expression systems (human kidney 293 cells and E. coli).
  • To assess the functional activity of E. coli-produced rTF.
  • To investigate the role of a specific cysteine residue in TF aggregation.

Main Methods:

  • Immunoaffinity purification of rTF using a TF-specific monoclonal antibody.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions.

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  • Functional assays including chromogenic and one-stage prothrombin time assays.
  • Site-directed mutagenesis to create a cysteine-to-serine variant (C245S).

    • Main Results:

    • 293 cell-derived rTF is glycosylated (45K apparent MW) and forms disulfide-bonded dimers.
    • E. coli-derived rTF (33K/35K MW) lacks glycosylation but is fully functional.
    • The C245S variant shows significantly reduced disulfide-linked aggregates after purification.
    • The C245S variant retains full functional activity in coagulation assays.

    Conclusions:

    • E. coli can be used to produce functional, non-glycosylated rTF.
    • Mutating cytoplasmic cysteine 245 reduces TF aggregation without compromising activity.
    • These findings facilitate the production of stable and active recombinant TF for further studies.