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Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli

Bio/Technology volume 7, pages 495–501 (1989)Cite this article

Abstract

Using different types of efficient Escherichia coli expression systems, we have been able to obtain levels of human tissue-type plasminogen activator (tPA) reaching at least five percent of total bacterial proteins. The natural precursor of tPA (pre-tPA) expressed in E. coli accumulates, most probably in the cytoplasm, partly due to the low efficiency of the tPA leader sequence as a signal for secretion into the E. coli periplasm. We also expressed mature tPA in an insoluble, aggregated form and treatments were developed that permitted significant renaturation of this protein from the lysis pellet. Further characterization and purification of the rena-tured tPA polypeptide strongly suggest that a fully active enzyme, very similar to the eukaryotic product can be obtained, despite the total lack of glycosylation.

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Author notes

  1. Paolo Sarmientos

    Present address: Dipartimento di Biologia Molecolare Farmitalia-Carlo Erba, Viale Bezzi 24, 20146, Milano, Italy

  2. Terence Cartwright: To whom correspondence should be addressed.

Authors and Affiliations

  1. Laboratoire de Génétique, Institut de Biotechnologie, Centre de Recherches de Vitry, Rhône-Poulenc Santé, B.P.14, 94403, Vitry sur Seine Cedex, France

    Patrice Denèfle & Jean-François Mayaux

  2. Laboratoire de Biochimie des Macromolécules, Institut de Biotechnologie, Centre de Recherches de Vitry, Rhône-Poulenc Santé, B.P.14, 94403, Vitry sur Seine Cedex, France

    Marc Duchesne, Janine Boiziau, Nadine Fromage, Nadine Delporte, Fabienne Parker, Yves Lelièvre & Terence Cartwright

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Sarmientos, P., Duchesne, M., Denèfle, P. et al. Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli. Nat Biotechnol 7, 495–501 (1989). https://doi.org/10.1038/nbt0589-495

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