Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli
Author: ["Paolo Sarmientos","Marc Duchesne","Patrice Denèfle","Janine Boiziau","Nadine Fromage","Nadine Delporte","Fabienne Parker","Yves Lelièvre","Jean-François Mayaux","Terence Cartwright"]
Publication: Bio/Technology
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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.
Cite this article
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
