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Related Experiment Videos

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector.

A M Furlong1, D R Thomsen, K R Marotti

  • 1Biotechnology, Upjohn Company, Kalamazoo, Michigan 49007.

Biotechnology and Applied Biochemistry
|October 1, 1988
PubMed
Summary

This study successfully produced active tissue plasminogen activator (TPA) using a baculovirus expression system in insect cells. The recombinant TPA showed comparable activity to standards, indicating potential for biotechnological applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Expression

Background:

  • Tissue plasminogen activator (TPA) is a crucial enzyme in fibrinolysis.
  • Efficient and scalable production of active TPA is essential for therapeutic and research applications.
  • Baculovirus expression systems offer a robust platform for recombinant protein production in insect cells.

Purpose of the Study:

  • To develop a baculovirus expression vector for producing recombinant human tissue plasminogen activator (TPA).
  • To characterize the activity, processing, and molecular properties of TPA expressed in insect cells.
  • To compare the recombinant TPA with a standard TPA preparation.

Main Methods:

  • Construction of a baculovirus expression vector with TPA cDNA under the polyhedrin promoter.

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  • Infection of insect cells and isolation of TPA from conditioned media.
  • Purification using metal chelate affinity and immunoaffinity chromatography.
  • Analysis by SDS-PAGE, N-terminal sequencing, and fibrin agar activity gels.

    • Main Results:

    • Active, two-chain TPA was secreted into the culture medium by infected insect cells.
    • N-terminal sequencing confirmed processing at the N-terminus, similar to mammalian cells.
    • Recombinant TPA exhibited comparable specific activity to Bowes melanoma TPA, though with lower fibrinogen stimulation.
    • Apparent molecular weight suggested less complex glycosylation compared to mammalian-derived TPA.

    Conclusions:

    • The baculovirus expression system is effective for producing functional human TPA in insect cells.
    • Insect cell-processed TPA demonstrates biological activity and correct N-terminal processing.
    • The findings support the potential of this system for large-scale TPA production.